[60]
DISCOVERY
OF GLUTAMINE
SYNTHETASE
CASCADE
793
approximately 32,000, thus yields a heterodimer that possesses a fully active catalytic center. Evidence from X-ray crystallographic studies will be required to define this active center and corroborate the proposed reaction mechanism, 21 in which residues from both subunits are thought to participate.
[60] D i s c o v e r y o f G l u t a m i n e S y n t h e t a s e C a s c a d e
By EARL R. STADTMAN Studies on the regulation of glutamine synthetase (GS) (glutamateammonia ligase) activity were initiated in 1964 when Clifford Woolfolk joined the Laboratory of Biochemistry, National Heart, Lung, and Blood Institute. Woolfolk had just finished his graduate work at the University of Washington under the direction of Helen Whitely and wanted to investigate the regulation of branched metabolic pathways. Specifically, he wanted to continue some studies on the regulation of aspartate metabolism which I had carried out while on sabbatical leave in Georges Cohen's laboratory at the Pasteur Institute several years earlier. Not wanting to compete with Georges Cohen on a problem that was initiated in his own laboratory, I encouraged Woolfolk to select another problem, and suggested that he examine metabolic maps to identify some enzymes which catalyze reactions whose products serve as substrates in the first step of two or more divergent biosynthetic pathways. In addition, the multifunctional enzyme should be one whose activity could be easily and quickly measured. Woolfolk came up with three suggestions; namely, glutamate dehydrogenase, glutamine synthetase, and phosphoribosylpyrophosphate synthetase. Preliminary studies with glutamate dehydrogenase in extracts of Escherichia coli failed to disclose any unusual regulatory characteristics, so Woolfolk turned his attention to glutamine synthetase. We were delighted to find that the activity of GS in extracts ofE. coli was partially inhibited by each of eight different metabolites: histidine, tryptophan, AMP, CTP, carbamyl-P, glucosamine 6-phosphate, alanine, and glycine. 1-3 All these were known to be end products of glutamine metaboi C. A. Woolfolk and E. R. Stadtman, Biochem. Biophys. Res. Commun. 17, 313 (1964). 2 C. A. Woolfolk and E. R. Stadtman, Arch. Biochem. Biophys. 118, 736 (1967). 3 C. A. Woolfolk, B. Shapiro, and E. R. Stadtman, Arch. Biochem. Biophys. 116, 177 (1966).
METHODS IN ENZYMOLOGY, VOL. 182
794
APPENDIX
[60]
lism. 4 Woolfolk showed that in addition to feedback inhibition, the level of GS in E. coli was also under rigorous feedback control. The intracellular concentration of the enzyme could be varied more than 20-fold by variations in the availability of nitrogen in the growth medium. 3 Cumulative Feedback Inhibition After establishing that E. coli GS is subject to multiple feedback inhibition, Woolfolk et al. 3 obtained homogeneous crystalline preparations of the enzyme. From detailed hydrodynamic5-7 and electron microscopic examination,8 it was established that the enzyme had a molecular weight of 600K and is composed of 12 identical subunits, arranged in 2 superimposed hexagonal arrays. 8 On the basis of extensive kinetic measurements, it was demonstrated that at high, nearly saturating concentrations, each one of the eight different feedback inhibitors was by itself able to inhibit only a fraction (10-60%) of the GS activity. However, the inhibition obtained with a combination of any two of the metabolites was greater than with either one alone, and as the number of metabolites present was increased, there was a progressive increase in the extent of inhibition. When all eight metabolites were added together, almost complete inhibition of the enzyme activity was obtained. L2 Although other interpretations were not rigorously excluded, it was deduced from this unusual behavior that each subunit of GS contains a separate binding site for each one of the eight different feedback effectors. This phenomenon was referred to as "cumulative feedback inhibition." L2 The existence of separate binding sites for alanine, glycine, AMP, CTP, histidine, tryptophan, and also for Damino acids was subsequently verified by direct binding studies 9 and by the results of ligand-binding measurements utilizing stopped flow fluorescence, m-m2calorimetric, 13A4and NMR IL~2techniques. 4 E. R. Stadtman, in " T h e Enzymology of Glutamine Metabolism" (S. Prusiner and E. R. Stadtman, eds.), p. 1. Academic Press, New York, 1973. 5 C. A. Woolfolk and E. R. Stadtman, Arch. Biochem. Biophys. 122, 174 (1967). 6 B. M. Shapiro and E. R. Stadtman, J. Biol. Chem. 242, 5069 (1967). 7 B. M. Shapiro and A. Ginsburg, Biochemistry 7, 2153 (1968). 8 R. C. Valentine, B. M. Shapiro, and E. R. Stadtman, Biochemistry 7, 2143 (1968). 9 A. Ginsburg, Biochemistry 8, 1726 (1969). 10 S. G. Rhee, P. B. Chock, and E. R. Stadtman, in "Frontiers of Biological Energetics" (P. L. Dutton, J. S. Leigh, and A. Scarpa, eds.), Vol. 1, p. 725. Academic Press, New York, 1978. H S. G. Rhee, J. J. Villafranca, P. B. Chock, and E. R. Stadtman, Biochem. Biophys. Res. Commun. 78, 244 (1970). 12 j. j. Villafranca, S. G. Rhee, and P. B. Chock, proc. Natl. Acad. Sci. U.S.A. 75, 1255 (1978). 13 p. D. Ross and A. Ginsburg, Biochemistry 8, 4690 (1969). 14 A. Shrake, R. Park, and A. Ginsburg, Biochemistry 17, 658 (1978).
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
795
Adenylylation of GS The studies on GS regulation took an unexpected turn when the first batch of purified enzyme was nearly exhausted and a second batch was prepared. Much to our consternation, enzyme from the second batch enzyme was not inhibited by either one of the four metabolites, tryptophan, CTP, AMP, or histidine. 15 In other respects, the old and new batches of enzyme appeared to be indistinguishable. They possessed identical hydrodynamic properties, circular dichroism (CD) spectrum, and amino acid composition. Efforts to normalize their responses to feedback inhibitors by a variety of treatments (exposure to heat, sulfhydryl reagents, ionic strength, 02, urea, etc.) were unsuccessful. It was therefore concluded that the difference in feedback inhibition patterns must reflect subtle differences in the properties of the cells from which the enzymes were isolated. Henry Kingdon therefore undertook a systematic investigation of the inhibition patterns of GS in extracts of various batches of E. coli which had been grown under different conditions. His efforts were rewarded by the finding that GS from nitrogen-starved cells was not inhibited by AMP, histidine, tryptophan, or CTP, whereas the enzyme from cells grown on a nitrogen-rich medium was inhibited by all of these metabolites. 15 The discrepancy in the behavior of enzyme preparations from different batches of cells was therefore attributable to variations in E. coli growth conditions. The molecular basis for their differences in feedback inhibition was eventually disclosed by a comparison of the ultraviolet absorption spectrum of highly purified preparations of both forms of enzyme.16 The spectrum of the enzyme from cells grown on a nitrogen-rich medium exhibited significantly higher absorbance in the region of 260 nm (Fig. 1). Indeed, the difference spectrum obtained when equal amounts of the two preparations were compared directly, one against the other, exhibited a peak at 260 nm (Fig. 1, inset), suggesting that the preparation from nitrogen-rich medium was associated with a purine derivative. The 260rim-absorbing material was evidently covalently bound to the protein since it was not released from the protein by gel filtration, exhaustive dialysis, treatment with charcoal or with acid ammonium sulfate, or by incubating with 0.3 M HCI or 0.3 M NaOH for 3 hr at 37°, or by precipitation with 15% perchloric acid. Upon treatment with snake venom phosphodiesterase (SVP), AMP was released from the protein and, coincidentally, the enzyme was converted to a form apparently identical to that isolated from nitrogen-starved cells. It was therefore obvious that the enzyme from ~5H. S. Kingdon and E. R. Stadtman, J. Bacteriol. 94, 949 (1967). ~6B. M. Shapiro, H. S., Kingdon, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 58, 642 (1967).
796
APPENDIX
5.0
7
..----.,,
[60]
o0 5 /
\
4.0
,~3.0
0
///"
/
i
I
I
v
\ 240 250 260 nm270280 290 300
1.0
250
260
270
280 ), nrn
290
300
310
FIG. 1. Ultraviolet absorption spectra of adenylylated and unadenylylated forms of glutamine synthetase. Dotted line, glutamine synthetase from cells grown in nitrogen-rich medium; solid line, glutamine synthetase from cells grown in nitrogen-limiting medium; inset, difference spectrum between the two forms. Extinction coefficients are expressed per mole of enzyme of M, 600,000.
nitrogen-rich cells contained an adenylic acid residue attached in phosphodiester linkage to some amino acid residue in the enzyme. To identify the site of attachment, the adenylylated enzyme was fragmented by treatment with pepsin and pronase. A decapeptide fragment containing the AMP moiety was isolated from the protease digest by means of charcoal adsorption and paper chromatography.~7 Of particular significance was the finding that the decapeptide contained no serine, threonine, or basic amino acid residues. In fact, a single tyrosine residue was the only residue in the peptide capable of forming a phosphodiester bond with AMP. That the AMP was attached to the tyrosine residue was verified by showing that removal of the AMP from the peptide by treatment with SVP led to exposure of a tyrosine hydroxyl group. This was established by showing that the characteristic increase in absorbance at 293 nm, which is associated with ionization of the tyrosyl hydroxyl group at pH 13, did not occur until after the AMP was released from the peptide by treatment with the diesterase. It was thus established that the catalytic activity and the susceptibility of E. coli GS to feedback regulation is mediated by the ade17 B. M. Shapiro and E. R. Stadtman, J. Biol. Chem. 243, 3769 (1968).
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
797
nylylation of a unique tyrosyl group in the enzyme. To our knowledge, this was the first example in which the esterification of a tryosine hydroxyl group was found to be implicated in enzyme regulation. Further comparison of the adenylylated and unadenylylated enzymes showed that the adenylylation of GS caused a shift in the pH activity profile and in the divalent cation specificity of the enzyme.15'16'lS-2°Thus, Mg 2+ was essential for the biosynthetic activity of the unadenylylated enzyme, whereas the adenylylated enzyme required Mn 2+. The pH optimum of the unadenylylated and adenylylated enzymes were 8.0 and 7.0, respectively. In contrast, Mn 2+ supported the ability of both enzyme forms to catalyze the nonphysiological y-glutamyl transfer reaction [reaction (1)] Glutamine + NH2OH
arsenate ADP
~ glutamylhydroxamate+ NH3
(1)
However, the pH-activity profiles of the two enzymes for catalysis of this reaction were significantly different. Under standardized assay conditions, Mg z+ could support transferase activity of the unadenylylated enzyme, but it selectively inhibited the MnE+-dependent transferase activity of the adenylylated enzyme. These characteristics were subsequently exploited in the development of a highly sensitive procedure for the estimation of the average state of adenylylation (h-) of GS in crude extracts. 19 Adenylyltransferase With the discovery that the activity and feedback control of GS is modulated by the esterification of a tyrosyl residue, it was obvious that E. coli contains an adenylyltransferase capable of catalyzing the adenylylation and deadenylylation of GS. The presence in E. coli extracts of an adenylyltransferase (ATase) that catalyzed transfer of the adenylyl group of ATP to GS was readily verified. When a protein fraction from E. coli extract was incubated with [14C]ATP and the unadenylylated form of GS, [~4C]AMP groups became attached to the GS and this adenylylation was accompanied by the expected changes in divalent cation specificity, pH-activity profile, and susceptibility to feedback inhibition. 18It was also evident from these studies that up to 12 adenylyl groups (1 per subunit) could be attached to each molecule of GS. The biosynthetic activity of the enzyme under physiological conditions is inversely proportional to the 18H. S. Kingdon,B. M. Shapiro, and E. R. Stadtman,Proc. Natl. Acad. Sci. U.S.A. 58, 1703 (1967). 19E. R. Stadtman,P. Z. Smyrniotis,J. N. Davis, and M. Wittenberger,Anal. Biochem. 95, 275 (1979). 2oA. Ginsburg,J. Yeh, S. B. Hennig,and M. D. Denton,Biochemistry 9, 633 (1970).
798
APPENDIX
[60]
average number (~) of adenylyl groups bound per molecule. Thus, the adenylylation of a particular subunit in the dodecameric enzyme leads to inactivation of that subunit only. The adenylyltransferase was subsequently purified to homogeneity by Ann Ginsburg in our laboratory 21 and also by Ebner et al. 22 in Holzer's laboratory in Freiburg. It was established that the adenylyltransferase is comprised of a single polypeptide chain ( M r : 130,000). It catalyzes the reversible reaction (2) 23 GS + 12 ATP ~ GS(AMP)n + 12 PPi
(2)
Relationship between Adenylyltransferase and Holzer's "Inactivation Enzyme" In 1966, Holzer and associates described an enzyme in E. coli that catalyzed the inactivation of GS. 24'z5 This "inactivase" required the presence of ATP and glutamine. In view of the fact that adenylylation of GS converts it from an Mga+-dependent form to an MnE÷-dependent form and also the fact that in Holzer's laboratory GS activity was always assayed in the presence of Mg z÷, i.e., under conditions where the adenylyled enzyme is inactive, it appeared likely that Holzer's "inactivase" was identical with Kingdon's adenylyltransferase. This was found to be the case. Further studies in Holzer's laboratory as well as our own confirmed that the "inactivase" and adenylyltransferase were one and the same enzyme. 16.26The adenylyltransferase was ultimately purified to homogeneity in both laboratories. 21'22 Furthermore, it was demonstrated in both laboratories that the interconversion of GS between adenylylated and unadenylylated forms occurs in vivo in response to shifts in the nutritional state of E. coli. Thus, as noted earlier, the adenylylated form is favored when E. coli is grown in a nitrogen-rich medium; but when cells are shifted from a nitrogen-rich to a nitrogen-poor medium, the GS was converted from the less active adenylylated form back to the more active unadenylylated form. 21 S. B. Hennig, W. B. Anderson, and A. Ginsburg, Proc. Natl. Acad. Sci. U.S.A. 67, 1761 (1970). 22 E. Ebner, D. Wolf, C. Gancedo, S. Elsiisser, and H. Holzer, Eur. J. Biochem. 14, 535 (1970). 23 M. Mantel and H. Holzer, Proc. Natl. Acad. Sci. U.S.A. 65, 660 (1970). 24 D. Mecke and H. Holzer, Biochim. Biophys. Acta 122, 341 (1966). D. Mecke, K. Wulff, K. Liess, and H. Holzer, Biochem. Biophys. Res. Commun. 24, 542 (1966). 26 K. Wulff, D. Mecke, and H. Holzer, Biochem. Biophys. Res. Commun. 28, 740 (1967).
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
799
Deadenylylation of Adenylylated GS An activity capable of catalyzing the removal of adenylyl groups from adenylylated GS was detected in crude extracts of E. coli. 27 By means of gel filtration, B. M. Shapiro resolved the extract into two protein fractions, PI and PII, both of which were required for deadenylylation activity. He established further that the deadenylylation reaction required the presence of UTP and a-ketoglutarate, and was greatly stimulated by orthophosphate or arsenate, and was strongly inhibited by glutamine.28 Upon further purification, it became evident that the P~ fraction contained a single adenylyltransferase whose ability to catalyze the adenylylation of GS on the one hand and the deadenylylation of adenylylated GS on the other was somehow specified by the PII protein and by the concentrations of ATP, UTP, Pi, a-ketoglutarate, and glutamine, z9 The role of inorganic orthophosphate in this system was clarified by the studies of Anderson and Stadtman, 3° showing that the deadenylylation of GS does not involve simple reversal of the adenylylation reaction [reaction (1) ] but rather involves a phosphorolytic cleavage of the adenylyltyrosine bond of adenylylated GS to form ADP and the unmodified form of GS [reaction (3) ]: GS • AMP + P ~
GS + ADP
(3)
The PI could not be replaced by PP~. However, inorganic arsenate was able to substitute for P~, in which case AMP was the product, presumably obtained by spontaneous hydrolysis of the A M P - A s intermediate. Uridylylation of Pn Protein Governs Activity of Adenylyltransferase The role of the P~ protein in the differential regulation of the adenylylation and deadenylylation reactions was further clarified by the studies of Brown et al.,31 showing that when the PI~ protein was incubated with the PI fraction in the presence of ATP, UTP, a-ketoglutarate, and Mn 2÷, it was converted to a form which after reisolation by gel filtration was able, in the absence of UTP, to stimulate the ability of the PI fraction to catalyze the deadenylylation of adenylylated GS. Coincidentally, modification of 27 B. M. Shapiro and E. R. Stadtman, Biochem. Biophys. Res. Commun. 30, 32 (1968). 2a B. M. Shapiro, Biochemistry 8, 659 (1969). 29 W. B. Anderson, S. B. Hennig, A. Ginsburg, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 67, 1417 (1970). 30 W. B. Anderson and E. R. Stadtman, Biochem. Biophys. Res. Commun. 41, 704 (1970). 31 M. S. Brown, A. Segal, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 68, 2949 (1971).
800
APPENDIX
[60]
Pn led to a decrease in its ability to stimulate the adenylylation of GS. The possibility that the PII was covalently modified by a uridine derivative was indicated by the further demonstration that after incubation with [y_32p, 14C]UTP under the above conditions, the reisolated PII contained J4C but no 32p. Moreover, upon treatment with SVD, the modified Pn lost its ability to stimulate the deadenylylation reaction. Based on the results of these preliminary experiments, it was suggested that the unmodified form of PII stimulates the ability of adenylyltransferase to catalyze the adenylylation of GS, whereas a uridine derivative of the PII protein is able to stimulate the deadenylylation of adenylylated GS. It was further suggested that a-ketoglutarate and ATP stimulate and that glutamine inhibits the ability of an enzyme in the Pi protein fraction to catalyze the modification of Pli. It remained to be determined whether the UTP-dependent modification of the Pll protein was catalyzed by the adenylyltransferase itself or by some other enzyme in the relatively impure PI protein fraction. This question was answered with the demonstration that homogeneous preparations of the adenylyltransferase were unable to catalyze the covalent modification of PII, and also by the subsequent studies of Mangum e t a1.,32 showing that by means of chromatography on DE-52 cellulose, the adenylyltransferase in the PI fraction could be separated from a protein fraction capable of catalyzing both the uridylylation of the eli protein and the deuridylylation of the UMP-PII conjugate. Whether the uridylylation and deuridylylation reactions were catalyzed by separate enzymes or by a single bifunctional uridylyltransferase (UTase) in the Pl fraction was not readily solved because instability of the UTase activity eluded its purification. Moreover, the ratio of uridylylation and deuridylylation activities of a given enzyme preparation could be altered by aging or by exposure to mild denaturing conditions. It was not until several years later that Garcia and Rhee 33 succeeded in obtaining an apparently homogenous protein preparation of UTase which possessed both uridylylating and deuridylylating activities. Furthermore, by working rapidly they showed that the ratio of both activities remained constant throughout the purification procedure. Final proof that both activities were properties of a single bifunctional enzyme was afforded by the demonstration that a single point mutation in the structural gene led to the loss in expression of both activities. Because the PII preparation used in these studies was still relatively impure, neither its structure nor the stoichiometry could be ascertained. 32j. H. Mangum, G. Magni, and E. R. Stadtman, Arch. Biochem. Biophys. 158, 514 (1973). 33E. Garcia and S. G. Rhee, J. Biol. Chem. 258, 2246(1983).
[60]
DISCOVERYOF GLUTAMINESYNTHETASECASCADE
801
Adler e t al. 34 finally succeeded in obtaining homogeneous preparations of the PII protein and demonstrated that it was a protein of about Mr 44,000 and was composed of four apparently identical subunits. Furthermore, when this PII preparation was incubated with UTP, ATP, a-ketoglutarate, Mg 2÷ or Mn 2÷, and a more highly purified preparation of the UTase, up to 4 mol of UMP (one per subunit) could be covalently bound to each mole of the Pn protein. From amino acid analysis, it was found that each subunit of the Pn protein contained two tyrosine residues. Upon treatment with trypsin, two different tyrosine-containing peptides were generated which were easily separated by two-dimensional electrophoresis on acrylamide gels. Prior to uridylylation of the Pn protein, the tyrosine residues in both of the peptides could be iodinated with 125Iin the presence of chloramineT. However, after uridylylation, the tyrosine residue in just one of the two peptides could be iodinated. Since a free hydroxyl group is essential for iodination of a tryosyl group, this indicated that the UMP group was attached to the hydroxyl group of just one of the two tyrosine residues in the PH subunit. This conclusion was verified by the further observation that cleavage of the UMP groups from uridylylated PH by treatment with snake venom phosphodiesterase resulted in the exposure of a stoichiometric amount of ionizable tyrosyl hydroxyl groups as disclosed by spectral analysis. It was thus established that, as in the adenylylation of GS, the uridylylation of the PII protein involves the covalent attachment of the mononucleotide to tyrosyl residues. The Bicyclic Cascade With the above observations, it was evident that the activity of GS is under the fine control of a cascade system composed of two tightly linked interconvertible enzyme/protein cycles, each of which is catalyzed by a bifunctional enzyme (Fig. 2). From detailed analyses of the enzymes in this cascade, it was demonstrated that the activity of GS is subject to regulation by over 40 metabolites. 35'36 Some of these reacted with GS directly, whereas others exerted their effects by interactions with one or both of the two bifunctional enzymes. Of these effectors, ot-ketoglutarate, glutamate, ATP, UTP, and Pi are of special significance. The latter three compounds serve as cosubstrates in the nucleotidylation/denucleotidylationreactions, whereas glutamine and a-ketoglutarate are allosteric effectors of the bi34S. Adler, D. Purich, and E. R. Stadtman,J. Biol. Chem. 250, 6264 (1975). 35E. G. Englemanand S. H. Francis, Arch. Biochem. Biophys. 191, 602 (1978). 36E. R. Stadtmanand P. B. Chock, Curr. Top. Cell. Regul. 13, 53 (1978).
80:2
APPENDIX
I-I=O
[60l
UMP
---PII(UMP)4 Gin ,,
UT
a--KG Pu'-I
PPIp"
II I I
UTP A
GS
T
~
Gin \
~
I
PPi
GS(AMPh= ATd o,--KG / .
A D P ~ P i I I ~J Fio. 2. The bicyclic cascade of glutamine synthetase regulation. Interrelationship between the uridylylation cycle and the adenylylation cycle, and the reciprocal controls of these interconversions by L-glutamine (Gln) and ~-ketoglutarate (~-KG) are shown; + indicates stimulation, - indicates inhibition. Abbreviations: GS, glutamine synthetase; PH, regulatory protein; AT~ and ATd, the adenylylation and deadenylylation sites, respectively, on the bifunctional adenylyltransferase; UR and UT, the deuridylylation and uridylylation sites, respectively, on the bifunctional uridylyltransferase. In the text UT and UR are referred to as UTu and UTd, respectively (cf. Fig. 4). functional e n z y m e s . Thus, it was found that glutamine inhibits and a - k e t o glutarate stimulates the ability o f adenylyltransferase to catalyze the Plld e p e n d e n t adenylylation of GS at the adenylylation site (ATa) of ATase, w h e r e a s each effector exerts an opposite effect on the capacity o f A T a s e to catalyze the deadenylylation o f GS at the deadenylylation site (ATd) of the e n z y m e . 18,27,28 Similarly, glutamine was found to inhibit the ability of uridylyltransferase to catalyze the uridylylation o f Pn at the UTu site of U T a s e , but to stimulate its ability to catalyze the deuridylylation of Pn" U M P at the UTd site. In contrast, it was established that or-
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
803
ketoglutarate stimulates the deuridylylation reaction. In addition, it was determined that ATP serves as an essential allosteric activator for the UTase-catalyzed uridylylation of Pn, and that nucleoside monophosphates (especially CMP) serve as negative effectors of the deuridylylation reaction.31-35 Steady-State Concept The discovery that GS activity is regulated by the esterification of a tyrosyl hydroxyl group in the enzyme followed by more than two decades the earlier demonstration that covalent modification of an enzyme constitutes a physiological mechanism for the regulation of key metabolic processes. The pioneering studies of Cod, Krebs, Fisher, and Lamer showed quite clearly that the phosphorylation of seryl hydroxyl groups on glycogen phosphorylase and glycogen synthetase provided a physiological mechanism whereby the synthesis and degradation of glycogen could be regulated with respect to one another. This gave rise to the popular notion that the covalent modification of an enzyme constituted a physiological " s w i t c h " by means of which the activity of the enzyme could be turned " o n " or " o f f " in response to metabolic demand. Despite its attractive simplicity, this concept unrealistically ignored the fact that in order to serve as metabolic switches, activities of the protein kinases and phosphoprotein phosphatases would have to be reciprocally regulated in an " a l l " or " n o n e " fashion. The alternative possibility that dynamic, cyclic interconversion of an enzyme between covalently modified and unmodified forms provided a means by which an enzyme could be gradually shifted from one level of activity to another was not seriously considered. However, this possibility became evident from studies of Segal et al., 37 showing that the fraction of GS subunits that could be adenylylated varied in response to changes in the levels of multiple metabolites that govern the activities of the cascade enzymes. When GS was incubated in a mixture containing arbitrary concentrations of ATP, UTP, Pi, a-ketoglutarate, glutamine, Mg 2+ and/or Mn 2+, and the two bifunctional enzymes (ATase and UTase), within a few minutes the level of adenylylation, ff (i.e., the average number of adenylylated subunits per GS molecule), reached a steady-state value. Moreover, a change in the concentration of any one of the five metabolites or Mn 2+ caused a shift in the steady-state level of adenylylation of GS, either to higher or lower values, depending on which of the metabolite concentrations was altered (Fig. 3). It was established further that after 37A. Segal, M. S. Brown, and E. R. Stadtman,Arch. Biochem. Biophys. 161, 319 (1974).
APPENDIX
804 14
12
f
[60]
Effecter Varied O
a-KG
0
10
Pi
,,~-KG ATP =-
0
I
0
I
20
I
I
40
A
I
I
60
I
I
80
I
I
100
I
0 2
,4.5 2
None
UTP
Jt
mM
2
&
Gin
0.1
E}--O~
MnCI2 Gin
1.25 0
I
120
I
I
140
MINUTES
FIG. 3. Effect of metabolite concentrations on the steady-state level of adenylylated subunits. The heavy line (filled squares) shows the change of n- with time when 95/xg of glutamine synthetase was incubated in a mixture containing 20 mM MgC12, 20 mM Pi, 1 mM ATP, 1 mM UTP, 15 mM a-ketoglutarate, 0.3 mM glutamine, and partially purified preparations of Pa, ATase (containing also UTase). The other curves illustrate the effect of changing the concentration of only one metabolite in the mixture, as indicated.
assuming a steady-state value of if, ATP continued to be decomposed. These experiments demonstrated that for a given metabolic condition, a dynamic steady state is established in which the rates of adenylylation and deadenylylation of GS are equal, and that the fractions of GS subunits that are adenylylated in this steady state, and hence the specific catalytic activity of the enzyme, are specified by the relative concentrations of positive and negative effectors (metabolites) that govern the activity of the cascade enzymes. Contrary to the physiological switch concept, it became evident that the metabolic interconversion of enzymes between covalently modified and unmodified forms is a dynamic process which facilitates continuous shifts in the catalytic activities of the enzyme commensurate with metabolic demand. They established further that the decomposition of ATP associated with the cyclic interconversion of enzymes between
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
805
modified and unmodified forms is the price the cell pays to achieve such fine cellular control. Theoretical Analysis of Cyclic Cascade Systems Prompted by the above results, a detailed theoretical analysis of cyclic cascade systems was undertaken in order to define more exactly how such cycles respond to changes in the concentrations of metabolites which affect the cascade enzymes. 36-39 To our surprise, this analysis revealed that interconvertible enzyme cascades are endowed with many unique regulatory capacities that had not been previously suspected. Thus it was demonstrated that interconvertible enzyme cascades can respond simultaneously to a very large number of both positive and negative allosteric effectors and thereby generate a multitude of fundamentally different regulatory patterns. These cascades are also capable of signal amplification, i.e., the concentration of a given allosteric effector that is able to provoke a large change in the level of covalently modified interconvertible enzyme can be orders of magnitude below the Km for the binding of that effector to the converter enzymes. Cyclic cascades may also serve as rate amplifiers; and therefore can facilitate a change from one steady-state level of covalent modification to another within the millisecond time range. In addition, interconvertible enzyme cascades are capable of generating a cooperative-type (sigmoidal) response to increasing concentrations of a given effector. Verification of Theoretical Predictions Whereas these studies had focused attention on the dynamics of interconvertible enzyme systems, the kind of detailed in vitro studies needed to verify predictions derived from the theoretical analysis of cyclic cascades was hampered by the instability of UTase, which had precluded the isolation of appreciable quantities of the enzyme. Nevertheless, because the primary function of UTase is to regulate the Pu/Pxr (UMP) ratio, it was evident that the effect of UTase to the overall adenylylation of GS could be mimicked by varying the mole fraction [Pn]/[Pn]+[Pn. UMP]. Therefore, a massive kinetic study was carried out to determine if the properties of cyclic cascades disclosed by the theoretical analysis could be verified. 4° 38 E. R. Stadtman and P. B. Chock, Proc. Natl. Acad. Sci. U.S.A. 74, 2761 (1978). 39 p. B. Chock and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 74, 2766 (1977). 4o S. G. Rhee, R. Park, P. B. Chock, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 75, 3138 (1978).
806
APPENDIX
[60]
To this end, it was necessary to determine experimentally the values of 28 different reaction constants needed to describe the theoretical model and, in addition, to determine how the steady-state level of GS adenylylation varied when GS is incubated with a highly purified preparation of ATase in the presence of various concentrations of glutamine and a-ketoglutarate and at a number of different mole fractions of Pu. Results of these studies confirmed that cyclic cascades are in fact endowed with all of the properties which were predicted by the theoretical considerations. Verification of these basic principles was obtained also from studies in which the state of adenylylation of GS was measured in permeabilized E. coli cells following their incubation in buffer containing variable concentrations of ATP, UTP, o~-ketoglutarate, glutamine, and Pi .41,42 Application of Molecular Biology Technology It is perhaps worth noting that the discovery and elucidation of the GS cascade as summarized in the above sections was achieved by the application of classical enzymological approaches and technologies. None of the modern techniques of molecular biology or of biochemical genetics was utilized. Indeed, it is questionable whether the application of these latter techniques would have helped or hindered progress in the elucidation of this remarkable cascade system. Nevertheless, as noted earlier, a quantitative study of this bicyclic cascade system was hampered by the instability of the UTase, which precluded its isolation as a homogeneous protein, and also by the fact that, except for GS, the intracellular concentrations of the cascade enzymes are relatively low, making it difficult to obtain sufficient quantities of homogeneous preparations needed for detailed studies of the bicyclic cascade in vitro. To overcome these problems, we resorted to the use of molecular biology approaches to obtain strains ofE. coli which produced 800-, 500-, and 70-fold more UTase, ATase, and PII protein, respectively, than the wild-type strain. 43,44 Using these strains, Rhee and colleagues obtained a substantial quantity of a homogeneous preparation of each one of the cascade enzymes. In a monumental e f f o r t , 45 they determined the values of 21 interaction constants that govern the protein/protein and protein/ 41 U. Mura and E. R. Stadtman, J. Biol. Chem. 256, 13014 (1981). 42 U. Mura, P. B. Chock, and E. R. Stadtman, J. Biol. Chem. 256, 13022 (1981). 43 S. G. Rhee, S. C. Park, and J. H. Koo, Curr. Top. Cell. Regul. 27, 221 (1985). 44 H. S. Son and S. G. Rhee, J. Biol. Chem. 262, 8690 (1987). 45 S. G. Rhee, W. G. Bang, S. C. Park, J. H. Koo, and K. H. Min, in "Dynamics of Soluble and Immobilized Enzymes" (P. B. Chock, L. Tsou, and C. Y. Huang, eds.), p. 128. Springer-Verlag, Amsterdam, 1987.
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
807
effector interactions which are involved in various steps of the bicyclic cascade. Then, in order to simulate in vitro the characteristics of the GS cascade, a series of experiments was carried out in which highly purified preparations of the cascade enzymes and the Pu protein were mixed together in amounts equivalent to those found in crude extracts ofE. coli. In this study, the effects of variations in the relative concentrations of glutamine and a-ketoglutarate were examined under three different conditions, namely: (1) in the presence ofUTP, Pi, ATP, UTase, and PII only (to mimic the operation of the uridylylation/deuridylylation cycle alone), (2) in the presence of ATase, ATP, and varying ratios of Pu and PII" UMP (to mimic operation of the adenylylation/deadenylylation cycle alone), and (3) in the presence of all substrates and cascade enzymes (to mimic the behavior of the coupled bicyclic cascade). Suffice it to say that the results of these studies confirmed in every important detail theoretical predictions of the cascade model, and illustrated directly the remarkable regulatory features of such cascades .45 It was evident from the results of these studies that interconvertible enzyme cascades serve as metabolic integration systems. By means of allosteric and substrate site interactions, the interconvertible enzymes are programmed to sense fluctuations in the concentrations of a multiplicity of metabolites. This leads to automatic adjustments in the specific activities and kinetic constants of the several cascade enzymes. Through this system the multiple inputs are integrated and registered as a single output, the fractional modification of the target enzyme, and thereby determines its specific catalytic activity. Role of UTase and Pu in Regulation of Glutamine Synthetase Formation In the initial studies on the regulation of GS activity, Woolfolk noted that the level of GS in E. coli was dependent on the availability of nitrogen in the culture medium. In the meantime, the mechanism that underlies the nitrogen control of GS synthesis was under extensive investigation in the laboratory of M a g a s a n i k 46'47 and K u s t u . 48'49 In both laboratories, a detailed genetic analysis of the GS cascade led eventually to the demonstration that UTase and the Pn protein are involved in the nitrogen control of GS levels. Such roles were suggested also by the studies of Rhee and 46 B. Magasanik, Annu. Rev. Genet. 16, 135 (1982). 47 B. Magasanik, TIBS 13, 475 (1988). 48 S. Kustu, K. Sei, and J. Keener, in "Regulation of Gene Expression" (I. R. Booth and C. F. Higgins, eds.), p. 139. Cambridge Univ. Press, London, 1986. 49 j. Keener, P. Wong, D. Popham, J. Wallis, and S. Kustu, in " R N A Polymerase and Regulation of Transcription" (W. S. Reznikoff, R. R. Burgess, J. E. Dahlberg, C. A. Gross, M. T. Record, Jr., and M. W. Wickens, eds.), p. 159. Elsevier, New York, 1987.
808
APPENDIX
[60]
j,s ~ m j'
GSactivity
HzO
"~
UMP
\ p]i_U ~~P I _UTd _ ~P,]]
PPi
ADP 10 P. ~ J'
UTP
ATP
~ PPi
HzO (~
Pi
N ~ I ~ N I
GS synthesis
ADP
ATP
FIG. 4. The cyclic cascade of glutamine synthetase (GS) regulation. Interrelationship between the uridylylation cycle, the adenylylation cycle, and the phosphorylation cycle; the reciprocal controls of these interconversions by L-glutamine (Gin) and a-ketoglutarate (a-KG) are shown; 0) indicates stimulation, O indicates inhibition. Abbreviations: GS, glutamine synthetase; PII, regulatory protein; Ata and ATd, adenylylation and deadenylylation sites, respectively, on the bifunctional adenylyltransferase; UTu and UTd, uridylylation and deuridylylation (uridylyl-removing) sites, respectively, on the bifunctional uridylyltransferase. NRI, glnG product also known as NTRC; NRuK and NRap, glnL product (also known as NTRB) catalyzing phosphorylation and dephosphorylation of NR~, respectively.
colleagues, showing that the repression of GS synthesis which occurs when E. coli is grown in a nitrogen-rich medium is regulated in part by the intracellular concentrations of Pzi, UTase, and ATase. 45The participation of Pu and UTase in the repression of GS synthesis was ultimately clarified by the elegant studies of Ninfa and Magasanik 5° and Keener and Kustu. 51 They showed that transcription of the structural gene for GS is under the control of several gene products, two of which, glnG (ntrC) and glnL (ntrB), are members of the gln operon. The product of the glnL gene (NRII) is a protein kinase that catalyzes the phosphorylation of the glnG so A. J. Ninfa and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A. 83, 5909 (1986). 51j. Keener and S. Kustu, Proc. Natl. Acad. Sci. U.S.A. 85, 4976 (1988).
[61]
ELONGATIONFACTORS
809
product (NRI) and thereby converts it to a form, NRI-P, which can activate glnA transcription. However, the cyclic interconversion of the glnG product between phosphorylated (NRI-P) and unphosphorylated (NRI) forms is dependent upon the concentration of the PII protein which stimulates the dephosphorylation of NRI-P. Whether the effect of Pn is to convert the NRu from a kinase to a phosphatase or serves only as an effector which together with NRII accelerates the spontaneous dephosphorylation of NRI-P appears unsettled. In any case, it became evident from these studies and those summarized above that the regulation of GS activity on the one hand and the regulation of GS formation on the other are tightly linked via interconversion of PII between its unmodified and uridylylated forms (Fig. 4). It is in fact the UTase which via allosteric interactions senses changes in the concentrations of a-ketoglutarate and glutamine and thereby dictates the steady-state levels of PII and PII"UMP; these in turn specify the activities of NRII and ATase, which in turn determine the steady-state levels of NRI-P and adenylylated GS, and hence the rate of GS synthesis and GS activity.
[61] D i s c o v e r y , R e s o l u t i o n , P u r i f i c a t i o n , a n d F u n c t i o n o f Elongation Factors By KIVIE MOLDAVE
In the early 1950s, most of the research on protein biosynthesis focused on the cellular components required for the incorporation of amino acids into proteins in cell-free extracts in vitro. Phil Siekevitz ~found that incubation of rat liver mitochondria, cytosol, and microsomes (fragments of endoplasmic reticulum membranes containing ribosomes, nonribosomal proteins, endogenous mRNA, lipids, etc.) accounted for most of the incorporation of amino acids observed when whole homogenates were used; mitochondrial oxidative phosphorylation was necessary. Zamecnik and Keller 2 obtained a more refined system that consisted of microsomes, a nondialyzable heat-labile fraction from the cytosol, and an ATPgenerating system; the energy requirements provided by mitochondrial oxidative phosphorylation were replaced by ATP and an ATP-generating P. S i e k e v i t z , J. Biol. Chem. 195, 549 (1952). 2 p. C. Z a m e c n i k and E. B. Keller, J. Biol. Chem. 2119, 337 (1954).
METHODS IN ENZYMOLOGY, VOL. 182
Copyright© 1990by AcademicPress, Inc. All rightsof reproductionin any form reserved.
DISCOVERY
OF GLUTAMINE
SYNTHETASE
CASCADE
793
approximately 32,000, thus yields a heterodimer that possesses a fully active catalytic center. Evidence from X-ray crystallographic studies will be required to define this active center and corroborate the proposed reaction mechanism, 21 in which residues from both subunits are thought to participate.
[60] D i s c o v e r y o f G l u t a m i n e S y n t h e t a s e C a s c a d e
By EARL R. STADTMAN Studies on the regulation of glutamine synthetase (GS) (glutamateammonia ligase) activity were initiated in 1964 when Clifford Woolfolk joined the Laboratory of Biochemistry, National Heart, Lung, and Blood Institute. Woolfolk had just finished his graduate work at the University of Washington under the direction of Helen Whitely and wanted to investigate the regulation of branched metabolic pathways. Specifically, he wanted to continue some studies on the regulation of aspartate metabolism which I had carried out while on sabbatical leave in Georges Cohen's laboratory at the Pasteur Institute several years earlier. Not wanting to compete with Georges Cohen on a problem that was initiated in his own laboratory, I encouraged Woolfolk to select another problem, and suggested that he examine metabolic maps to identify some enzymes which catalyze reactions whose products serve as substrates in the first step of two or more divergent biosynthetic pathways. In addition, the multifunctional enzyme should be one whose activity could be easily and quickly measured. Woolfolk came up with three suggestions; namely, glutamate dehydrogenase, glutamine synthetase, and phosphoribosylpyrophosphate synthetase. Preliminary studies with glutamate dehydrogenase in extracts of Escherichia coli failed to disclose any unusual regulatory characteristics, so Woolfolk turned his attention to glutamine synthetase. We were delighted to find that the activity of GS in extracts ofE. coli was partially inhibited by each of eight different metabolites: histidine, tryptophan, AMP, CTP, carbamyl-P, glucosamine 6-phosphate, alanine, and glycine. 1-3 All these were known to be end products of glutamine metaboi C. A. Woolfolk and E. R. Stadtman, Biochem. Biophys. Res. Commun. 17, 313 (1964). 2 C. A. Woolfolk and E. R. Stadtman, Arch. Biochem. Biophys. 118, 736 (1967). 3 C. A. Woolfolk, B. Shapiro, and E. R. Stadtman, Arch. Biochem. Biophys. 116, 177 (1966).
METHODS IN ENZYMOLOGY, VOL. 182
794
APPENDIX
[60]
lism. 4 Woolfolk showed that in addition to feedback inhibition, the level of GS in E. coli was also under rigorous feedback control. The intracellular concentration of the enzyme could be varied more than 20-fold by variations in the availability of nitrogen in the growth medium. 3 Cumulative Feedback Inhibition After establishing that E. coli GS is subject to multiple feedback inhibition, Woolfolk et al. 3 obtained homogeneous crystalline preparations of the enzyme. From detailed hydrodynamic5-7 and electron microscopic examination,8 it was established that the enzyme had a molecular weight of 600K and is composed of 12 identical subunits, arranged in 2 superimposed hexagonal arrays. 8 On the basis of extensive kinetic measurements, it was demonstrated that at high, nearly saturating concentrations, each one of the eight different feedback inhibitors was by itself able to inhibit only a fraction (10-60%) of the GS activity. However, the inhibition obtained with a combination of any two of the metabolites was greater than with either one alone, and as the number of metabolites present was increased, there was a progressive increase in the extent of inhibition. When all eight metabolites were added together, almost complete inhibition of the enzyme activity was obtained. L2 Although other interpretations were not rigorously excluded, it was deduced from this unusual behavior that each subunit of GS contains a separate binding site for each one of the eight different feedback effectors. This phenomenon was referred to as "cumulative feedback inhibition." L2 The existence of separate binding sites for alanine, glycine, AMP, CTP, histidine, tryptophan, and also for Damino acids was subsequently verified by direct binding studies 9 and by the results of ligand-binding measurements utilizing stopped flow fluorescence, m-m2calorimetric, 13A4and NMR IL~2techniques. 4 E. R. Stadtman, in " T h e Enzymology of Glutamine Metabolism" (S. Prusiner and E. R. Stadtman, eds.), p. 1. Academic Press, New York, 1973. 5 C. A. Woolfolk and E. R. Stadtman, Arch. Biochem. Biophys. 122, 174 (1967). 6 B. M. Shapiro and E. R. Stadtman, J. Biol. Chem. 242, 5069 (1967). 7 B. M. Shapiro and A. Ginsburg, Biochemistry 7, 2153 (1968). 8 R. C. Valentine, B. M. Shapiro, and E. R. Stadtman, Biochemistry 7, 2143 (1968). 9 A. Ginsburg, Biochemistry 8, 1726 (1969). 10 S. G. Rhee, P. B. Chock, and E. R. Stadtman, in "Frontiers of Biological Energetics" (P. L. Dutton, J. S. Leigh, and A. Scarpa, eds.), Vol. 1, p. 725. Academic Press, New York, 1978. H S. G. Rhee, J. J. Villafranca, P. B. Chock, and E. R. Stadtman, Biochem. Biophys. Res. Commun. 78, 244 (1970). 12 j. j. Villafranca, S. G. Rhee, and P. B. Chock, proc. Natl. Acad. Sci. U.S.A. 75, 1255 (1978). 13 p. D. Ross and A. Ginsburg, Biochemistry 8, 4690 (1969). 14 A. Shrake, R. Park, and A. Ginsburg, Biochemistry 17, 658 (1978).
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
795
Adenylylation of GS The studies on GS regulation took an unexpected turn when the first batch of purified enzyme was nearly exhausted and a second batch was prepared. Much to our consternation, enzyme from the second batch enzyme was not inhibited by either one of the four metabolites, tryptophan, CTP, AMP, or histidine. 15 In other respects, the old and new batches of enzyme appeared to be indistinguishable. They possessed identical hydrodynamic properties, circular dichroism (CD) spectrum, and amino acid composition. Efforts to normalize their responses to feedback inhibitors by a variety of treatments (exposure to heat, sulfhydryl reagents, ionic strength, 02, urea, etc.) were unsuccessful. It was therefore concluded that the difference in feedback inhibition patterns must reflect subtle differences in the properties of the cells from which the enzymes were isolated. Henry Kingdon therefore undertook a systematic investigation of the inhibition patterns of GS in extracts of various batches of E. coli which had been grown under different conditions. His efforts were rewarded by the finding that GS from nitrogen-starved cells was not inhibited by AMP, histidine, tryptophan, or CTP, whereas the enzyme from cells grown on a nitrogen-rich medium was inhibited by all of these metabolites. 15 The discrepancy in the behavior of enzyme preparations from different batches of cells was therefore attributable to variations in E. coli growth conditions. The molecular basis for their differences in feedback inhibition was eventually disclosed by a comparison of the ultraviolet absorption spectrum of highly purified preparations of both forms of enzyme.16 The spectrum of the enzyme from cells grown on a nitrogen-rich medium exhibited significantly higher absorbance in the region of 260 nm (Fig. 1). Indeed, the difference spectrum obtained when equal amounts of the two preparations were compared directly, one against the other, exhibited a peak at 260 nm (Fig. 1, inset), suggesting that the preparation from nitrogen-rich medium was associated with a purine derivative. The 260rim-absorbing material was evidently covalently bound to the protein since it was not released from the protein by gel filtration, exhaustive dialysis, treatment with charcoal or with acid ammonium sulfate, or by incubating with 0.3 M HCI or 0.3 M NaOH for 3 hr at 37°, or by precipitation with 15% perchloric acid. Upon treatment with snake venom phosphodiesterase (SVP), AMP was released from the protein and, coincidentally, the enzyme was converted to a form apparently identical to that isolated from nitrogen-starved cells. It was therefore obvious that the enzyme from ~5H. S. Kingdon and E. R. Stadtman, J. Bacteriol. 94, 949 (1967). ~6B. M. Shapiro, H. S., Kingdon, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 58, 642 (1967).
796
APPENDIX
5.0
7
..----.,,
[60]
o0 5 /
\
4.0
,~3.0
0
///"
/
i
I
I
v
\ 240 250 260 nm270280 290 300
1.0
250
260
270
280 ), nrn
290
300
310
FIG. 1. Ultraviolet absorption spectra of adenylylated and unadenylylated forms of glutamine synthetase. Dotted line, glutamine synthetase from cells grown in nitrogen-rich medium; solid line, glutamine synthetase from cells grown in nitrogen-limiting medium; inset, difference spectrum between the two forms. Extinction coefficients are expressed per mole of enzyme of M, 600,000.
nitrogen-rich cells contained an adenylic acid residue attached in phosphodiester linkage to some amino acid residue in the enzyme. To identify the site of attachment, the adenylylated enzyme was fragmented by treatment with pepsin and pronase. A decapeptide fragment containing the AMP moiety was isolated from the protease digest by means of charcoal adsorption and paper chromatography.~7 Of particular significance was the finding that the decapeptide contained no serine, threonine, or basic amino acid residues. In fact, a single tyrosine residue was the only residue in the peptide capable of forming a phosphodiester bond with AMP. That the AMP was attached to the tyrosine residue was verified by showing that removal of the AMP from the peptide by treatment with SVP led to exposure of a tyrosine hydroxyl group. This was established by showing that the characteristic increase in absorbance at 293 nm, which is associated with ionization of the tyrosyl hydroxyl group at pH 13, did not occur until after the AMP was released from the peptide by treatment with the diesterase. It was thus established that the catalytic activity and the susceptibility of E. coli GS to feedback regulation is mediated by the ade17 B. M. Shapiro and E. R. Stadtman, J. Biol. Chem. 243, 3769 (1968).
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
797
nylylation of a unique tyrosyl group in the enzyme. To our knowledge, this was the first example in which the esterification of a tryosine hydroxyl group was found to be implicated in enzyme regulation. Further comparison of the adenylylated and unadenylylated enzymes showed that the adenylylation of GS caused a shift in the pH activity profile and in the divalent cation specificity of the enzyme.15'16'lS-2°Thus, Mg 2+ was essential for the biosynthetic activity of the unadenylylated enzyme, whereas the adenylylated enzyme required Mn 2+. The pH optimum of the unadenylylated and adenylylated enzymes were 8.0 and 7.0, respectively. In contrast, Mn 2+ supported the ability of both enzyme forms to catalyze the nonphysiological y-glutamyl transfer reaction [reaction (1)] Glutamine + NH2OH
arsenate ADP
~ glutamylhydroxamate+ NH3
(1)
However, the pH-activity profiles of the two enzymes for catalysis of this reaction were significantly different. Under standardized assay conditions, Mg z+ could support transferase activity of the unadenylylated enzyme, but it selectively inhibited the MnE+-dependent transferase activity of the adenylylated enzyme. These characteristics were subsequently exploited in the development of a highly sensitive procedure for the estimation of the average state of adenylylation (h-) of GS in crude extracts. 19 Adenylyltransferase With the discovery that the activity and feedback control of GS is modulated by the esterification of a tyrosyl residue, it was obvious that E. coli contains an adenylyltransferase capable of catalyzing the adenylylation and deadenylylation of GS. The presence in E. coli extracts of an adenylyltransferase (ATase) that catalyzed transfer of the adenylyl group of ATP to GS was readily verified. When a protein fraction from E. coli extract was incubated with [14C]ATP and the unadenylylated form of GS, [~4C]AMP groups became attached to the GS and this adenylylation was accompanied by the expected changes in divalent cation specificity, pH-activity profile, and susceptibility to feedback inhibition. 18It was also evident from these studies that up to 12 adenylyl groups (1 per subunit) could be attached to each molecule of GS. The biosynthetic activity of the enzyme under physiological conditions is inversely proportional to the 18H. S. Kingdon,B. M. Shapiro, and E. R. Stadtman,Proc. Natl. Acad. Sci. U.S.A. 58, 1703 (1967). 19E. R. Stadtman,P. Z. Smyrniotis,J. N. Davis, and M. Wittenberger,Anal. Biochem. 95, 275 (1979). 2oA. Ginsburg,J. Yeh, S. B. Hennig,and M. D. Denton,Biochemistry 9, 633 (1970).
798
APPENDIX
[60]
average number (~) of adenylyl groups bound per molecule. Thus, the adenylylation of a particular subunit in the dodecameric enzyme leads to inactivation of that subunit only. The adenylyltransferase was subsequently purified to homogeneity by Ann Ginsburg in our laboratory 21 and also by Ebner et al. 22 in Holzer's laboratory in Freiburg. It was established that the adenylyltransferase is comprised of a single polypeptide chain ( M r : 130,000). It catalyzes the reversible reaction (2) 23 GS + 12 ATP ~ GS(AMP)n + 12 PPi
(2)
Relationship between Adenylyltransferase and Holzer's "Inactivation Enzyme" In 1966, Holzer and associates described an enzyme in E. coli that catalyzed the inactivation of GS. 24'z5 This "inactivase" required the presence of ATP and glutamine. In view of the fact that adenylylation of GS converts it from an Mga+-dependent form to an MnE÷-dependent form and also the fact that in Holzer's laboratory GS activity was always assayed in the presence of Mg z÷, i.e., under conditions where the adenylyled enzyme is inactive, it appeared likely that Holzer's "inactivase" was identical with Kingdon's adenylyltransferase. This was found to be the case. Further studies in Holzer's laboratory as well as our own confirmed that the "inactivase" and adenylyltransferase were one and the same enzyme. 16.26The adenylyltransferase was ultimately purified to homogeneity in both laboratories. 21'22 Furthermore, it was demonstrated in both laboratories that the interconversion of GS between adenylylated and unadenylylated forms occurs in vivo in response to shifts in the nutritional state of E. coli. Thus, as noted earlier, the adenylylated form is favored when E. coli is grown in a nitrogen-rich medium; but when cells are shifted from a nitrogen-rich to a nitrogen-poor medium, the GS was converted from the less active adenylylated form back to the more active unadenylylated form. 21 S. B. Hennig, W. B. Anderson, and A. Ginsburg, Proc. Natl. Acad. Sci. U.S.A. 67, 1761 (1970). 22 E. Ebner, D. Wolf, C. Gancedo, S. Elsiisser, and H. Holzer, Eur. J. Biochem. 14, 535 (1970). 23 M. Mantel and H. Holzer, Proc. Natl. Acad. Sci. U.S.A. 65, 660 (1970). 24 D. Mecke and H. Holzer, Biochim. Biophys. Acta 122, 341 (1966). D. Mecke, K. Wulff, K. Liess, and H. Holzer, Biochem. Biophys. Res. Commun. 24, 542 (1966). 26 K. Wulff, D. Mecke, and H. Holzer, Biochem. Biophys. Res. Commun. 28, 740 (1967).
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
799
Deadenylylation of Adenylylated GS An activity capable of catalyzing the removal of adenylyl groups from adenylylated GS was detected in crude extracts of E. coli. 27 By means of gel filtration, B. M. Shapiro resolved the extract into two protein fractions, PI and PII, both of which were required for deadenylylation activity. He established further that the deadenylylation reaction required the presence of UTP and a-ketoglutarate, and was greatly stimulated by orthophosphate or arsenate, and was strongly inhibited by glutamine.28 Upon further purification, it became evident that the P~ fraction contained a single adenylyltransferase whose ability to catalyze the adenylylation of GS on the one hand and the deadenylylation of adenylylated GS on the other was somehow specified by the PII protein and by the concentrations of ATP, UTP, Pi, a-ketoglutarate, and glutamine, z9 The role of inorganic orthophosphate in this system was clarified by the studies of Anderson and Stadtman, 3° showing that the deadenylylation of GS does not involve simple reversal of the adenylylation reaction [reaction (1) ] but rather involves a phosphorolytic cleavage of the adenylyltyrosine bond of adenylylated GS to form ADP and the unmodified form of GS [reaction (3) ]: GS • AMP + P ~
GS + ADP
(3)
The PI could not be replaced by PP~. However, inorganic arsenate was able to substitute for P~, in which case AMP was the product, presumably obtained by spontaneous hydrolysis of the A M P - A s intermediate. Uridylylation of Pn Protein Governs Activity of Adenylyltransferase The role of the P~ protein in the differential regulation of the adenylylation and deadenylylation reactions was further clarified by the studies of Brown et al.,31 showing that when the PI~ protein was incubated with the PI fraction in the presence of ATP, UTP, a-ketoglutarate, and Mn 2÷, it was converted to a form which after reisolation by gel filtration was able, in the absence of UTP, to stimulate the ability of the PI fraction to catalyze the deadenylylation of adenylylated GS. Coincidentally, modification of 27 B. M. Shapiro and E. R. Stadtman, Biochem. Biophys. Res. Commun. 30, 32 (1968). 2a B. M. Shapiro, Biochemistry 8, 659 (1969). 29 W. B. Anderson, S. B. Hennig, A. Ginsburg, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 67, 1417 (1970). 30 W. B. Anderson and E. R. Stadtman, Biochem. Biophys. Res. Commun. 41, 704 (1970). 31 M. S. Brown, A. Segal, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 68, 2949 (1971).
800
APPENDIX
[60]
Pn led to a decrease in its ability to stimulate the adenylylation of GS. The possibility that the PII was covalently modified by a uridine derivative was indicated by the further demonstration that after incubation with [y_32p, 14C]UTP under the above conditions, the reisolated PII contained J4C but no 32p. Moreover, upon treatment with SVD, the modified Pn lost its ability to stimulate the deadenylylation reaction. Based on the results of these preliminary experiments, it was suggested that the unmodified form of PII stimulates the ability of adenylyltransferase to catalyze the adenylylation of GS, whereas a uridine derivative of the PII protein is able to stimulate the deadenylylation of adenylylated GS. It was further suggested that a-ketoglutarate and ATP stimulate and that glutamine inhibits the ability of an enzyme in the Pi protein fraction to catalyze the modification of Pli. It remained to be determined whether the UTP-dependent modification of the Pll protein was catalyzed by the adenylyltransferase itself or by some other enzyme in the relatively impure PI protein fraction. This question was answered with the demonstration that homogeneous preparations of the adenylyltransferase were unable to catalyze the covalent modification of PII, and also by the subsequent studies of Mangum e t a1.,32 showing that by means of chromatography on DE-52 cellulose, the adenylyltransferase in the PI fraction could be separated from a protein fraction capable of catalyzing both the uridylylation of the eli protein and the deuridylylation of the UMP-PII conjugate. Whether the uridylylation and deuridylylation reactions were catalyzed by separate enzymes or by a single bifunctional uridylyltransferase (UTase) in the Pl fraction was not readily solved because instability of the UTase activity eluded its purification. Moreover, the ratio of uridylylation and deuridylylation activities of a given enzyme preparation could be altered by aging or by exposure to mild denaturing conditions. It was not until several years later that Garcia and Rhee 33 succeeded in obtaining an apparently homogenous protein preparation of UTase which possessed both uridylylating and deuridylylating activities. Furthermore, by working rapidly they showed that the ratio of both activities remained constant throughout the purification procedure. Final proof that both activities were properties of a single bifunctional enzyme was afforded by the demonstration that a single point mutation in the structural gene led to the loss in expression of both activities. Because the PII preparation used in these studies was still relatively impure, neither its structure nor the stoichiometry could be ascertained. 32j. H. Mangum, G. Magni, and E. R. Stadtman, Arch. Biochem. Biophys. 158, 514 (1973). 33E. Garcia and S. G. Rhee, J. Biol. Chem. 258, 2246(1983).
[60]
DISCOVERYOF GLUTAMINESYNTHETASECASCADE
801
Adler e t al. 34 finally succeeded in obtaining homogeneous preparations of the PII protein and demonstrated that it was a protein of about Mr 44,000 and was composed of four apparently identical subunits. Furthermore, when this PII preparation was incubated with UTP, ATP, a-ketoglutarate, Mg 2÷ or Mn 2÷, and a more highly purified preparation of the UTase, up to 4 mol of UMP (one per subunit) could be covalently bound to each mole of the Pn protein. From amino acid analysis, it was found that each subunit of the Pn protein contained two tyrosine residues. Upon treatment with trypsin, two different tyrosine-containing peptides were generated which were easily separated by two-dimensional electrophoresis on acrylamide gels. Prior to uridylylation of the Pn protein, the tyrosine residues in both of the peptides could be iodinated with 125Iin the presence of chloramineT. However, after uridylylation, the tyrosine residue in just one of the two peptides could be iodinated. Since a free hydroxyl group is essential for iodination of a tryosyl group, this indicated that the UMP group was attached to the hydroxyl group of just one of the two tyrosine residues in the PH subunit. This conclusion was verified by the further observation that cleavage of the UMP groups from uridylylated PH by treatment with snake venom phosphodiesterase resulted in the exposure of a stoichiometric amount of ionizable tyrosyl hydroxyl groups as disclosed by spectral analysis. It was thus established that, as in the adenylylation of GS, the uridylylation of the PII protein involves the covalent attachment of the mononucleotide to tyrosyl residues. The Bicyclic Cascade With the above observations, it was evident that the activity of GS is under the fine control of a cascade system composed of two tightly linked interconvertible enzyme/protein cycles, each of which is catalyzed by a bifunctional enzyme (Fig. 2). From detailed analyses of the enzymes in this cascade, it was demonstrated that the activity of GS is subject to regulation by over 40 metabolites. 35'36 Some of these reacted with GS directly, whereas others exerted their effects by interactions with one or both of the two bifunctional enzymes. Of these effectors, ot-ketoglutarate, glutamate, ATP, UTP, and Pi are of special significance. The latter three compounds serve as cosubstrates in the nucleotidylation/denucleotidylationreactions, whereas glutamine and a-ketoglutarate are allosteric effectors of the bi34S. Adler, D. Purich, and E. R. Stadtman,J. Biol. Chem. 250, 6264 (1975). 35E. G. Englemanand S. H. Francis, Arch. Biochem. Biophys. 191, 602 (1978). 36E. R. Stadtmanand P. B. Chock, Curr. Top. Cell. Regul. 13, 53 (1978).
80:2
APPENDIX
I-I=O
[60l
UMP
---PII(UMP)4 Gin ,,
UT
a--KG Pu'-I
PPIp"
II I I
UTP A
GS
T
~
Gin \
~
I
PPi
GS(AMPh= ATd o,--KG / .
A D P ~ P i I I ~J Fio. 2. The bicyclic cascade of glutamine synthetase regulation. Interrelationship between the uridylylation cycle and the adenylylation cycle, and the reciprocal controls of these interconversions by L-glutamine (Gln) and ~-ketoglutarate (~-KG) are shown; + indicates stimulation, - indicates inhibition. Abbreviations: GS, glutamine synthetase; PH, regulatory protein; AT~ and ATd, the adenylylation and deadenylylation sites, respectively, on the bifunctional adenylyltransferase; UR and UT, the deuridylylation and uridylylation sites, respectively, on the bifunctional uridylyltransferase. In the text UT and UR are referred to as UTu and UTd, respectively (cf. Fig. 4). functional e n z y m e s . Thus, it was found that glutamine inhibits and a - k e t o glutarate stimulates the ability o f adenylyltransferase to catalyze the Plld e p e n d e n t adenylylation of GS at the adenylylation site (ATa) of ATase, w h e r e a s each effector exerts an opposite effect on the capacity o f A T a s e to catalyze the deadenylylation o f GS at the deadenylylation site (ATd) of the e n z y m e . 18,27,28 Similarly, glutamine was found to inhibit the ability of uridylyltransferase to catalyze the uridylylation o f Pn at the UTu site of U T a s e , but to stimulate its ability to catalyze the deuridylylation of Pn" U M P at the UTd site. In contrast, it was established that or-
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
803
ketoglutarate stimulates the deuridylylation reaction. In addition, it was determined that ATP serves as an essential allosteric activator for the UTase-catalyzed uridylylation of Pn, and that nucleoside monophosphates (especially CMP) serve as negative effectors of the deuridylylation reaction.31-35 Steady-State Concept The discovery that GS activity is regulated by the esterification of a tyrosyl hydroxyl group in the enzyme followed by more than two decades the earlier demonstration that covalent modification of an enzyme constitutes a physiological mechanism for the regulation of key metabolic processes. The pioneering studies of Cod, Krebs, Fisher, and Lamer showed quite clearly that the phosphorylation of seryl hydroxyl groups on glycogen phosphorylase and glycogen synthetase provided a physiological mechanism whereby the synthesis and degradation of glycogen could be regulated with respect to one another. This gave rise to the popular notion that the covalent modification of an enzyme constituted a physiological " s w i t c h " by means of which the activity of the enzyme could be turned " o n " or " o f f " in response to metabolic demand. Despite its attractive simplicity, this concept unrealistically ignored the fact that in order to serve as metabolic switches, activities of the protein kinases and phosphoprotein phosphatases would have to be reciprocally regulated in an " a l l " or " n o n e " fashion. The alternative possibility that dynamic, cyclic interconversion of an enzyme between covalently modified and unmodified forms provided a means by which an enzyme could be gradually shifted from one level of activity to another was not seriously considered. However, this possibility became evident from studies of Segal et al., 37 showing that the fraction of GS subunits that could be adenylylated varied in response to changes in the levels of multiple metabolites that govern the activities of the cascade enzymes. When GS was incubated in a mixture containing arbitrary concentrations of ATP, UTP, Pi, a-ketoglutarate, glutamine, Mg 2+ and/or Mn 2+, and the two bifunctional enzymes (ATase and UTase), within a few minutes the level of adenylylation, ff (i.e., the average number of adenylylated subunits per GS molecule), reached a steady-state value. Moreover, a change in the concentration of any one of the five metabolites or Mn 2+ caused a shift in the steady-state level of adenylylation of GS, either to higher or lower values, depending on which of the metabolite concentrations was altered (Fig. 3). It was established further that after 37A. Segal, M. S. Brown, and E. R. Stadtman,Arch. Biochem. Biophys. 161, 319 (1974).
APPENDIX
804 14
12
f
[60]
Effecter Varied O
a-KG
0
10
Pi
,,~-KG ATP =-
0
I
0
I
20
I
I
40
A
I
I
60
I
I
80
I
I
100
I
0 2
,4.5 2
None
UTP
Jt
mM
2
&
Gin
0.1
E}--O~
MnCI2 Gin
1.25 0
I
120
I
I
140
MINUTES
FIG. 3. Effect of metabolite concentrations on the steady-state level of adenylylated subunits. The heavy line (filled squares) shows the change of n- with time when 95/xg of glutamine synthetase was incubated in a mixture containing 20 mM MgC12, 20 mM Pi, 1 mM ATP, 1 mM UTP, 15 mM a-ketoglutarate, 0.3 mM glutamine, and partially purified preparations of Pa, ATase (containing also UTase). The other curves illustrate the effect of changing the concentration of only one metabolite in the mixture, as indicated.
assuming a steady-state value of if, ATP continued to be decomposed. These experiments demonstrated that for a given metabolic condition, a dynamic steady state is established in which the rates of adenylylation and deadenylylation of GS are equal, and that the fractions of GS subunits that are adenylylated in this steady state, and hence the specific catalytic activity of the enzyme, are specified by the relative concentrations of positive and negative effectors (metabolites) that govern the activity of the cascade enzymes. Contrary to the physiological switch concept, it became evident that the metabolic interconversion of enzymes between covalently modified and unmodified forms is a dynamic process which facilitates continuous shifts in the catalytic activities of the enzyme commensurate with metabolic demand. They established further that the decomposition of ATP associated with the cyclic interconversion of enzymes between
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
805
modified and unmodified forms is the price the cell pays to achieve such fine cellular control. Theoretical Analysis of Cyclic Cascade Systems Prompted by the above results, a detailed theoretical analysis of cyclic cascade systems was undertaken in order to define more exactly how such cycles respond to changes in the concentrations of metabolites which affect the cascade enzymes. 36-39 To our surprise, this analysis revealed that interconvertible enzyme cascades are endowed with many unique regulatory capacities that had not been previously suspected. Thus it was demonstrated that interconvertible enzyme cascades can respond simultaneously to a very large number of both positive and negative allosteric effectors and thereby generate a multitude of fundamentally different regulatory patterns. These cascades are also capable of signal amplification, i.e., the concentration of a given allosteric effector that is able to provoke a large change in the level of covalently modified interconvertible enzyme can be orders of magnitude below the Km for the binding of that effector to the converter enzymes. Cyclic cascades may also serve as rate amplifiers; and therefore can facilitate a change from one steady-state level of covalent modification to another within the millisecond time range. In addition, interconvertible enzyme cascades are capable of generating a cooperative-type (sigmoidal) response to increasing concentrations of a given effector. Verification of Theoretical Predictions Whereas these studies had focused attention on the dynamics of interconvertible enzyme systems, the kind of detailed in vitro studies needed to verify predictions derived from the theoretical analysis of cyclic cascades was hampered by the instability of UTase, which had precluded the isolation of appreciable quantities of the enzyme. Nevertheless, because the primary function of UTase is to regulate the Pu/Pxr (UMP) ratio, it was evident that the effect of UTase to the overall adenylylation of GS could be mimicked by varying the mole fraction [Pn]/[Pn]+[Pn. UMP]. Therefore, a massive kinetic study was carried out to determine if the properties of cyclic cascades disclosed by the theoretical analysis could be verified. 4° 38 E. R. Stadtman and P. B. Chock, Proc. Natl. Acad. Sci. U.S.A. 74, 2761 (1978). 39 p. B. Chock and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 74, 2766 (1977). 4o S. G. Rhee, R. Park, P. B. Chock, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 75, 3138 (1978).
806
APPENDIX
[60]
To this end, it was necessary to determine experimentally the values of 28 different reaction constants needed to describe the theoretical model and, in addition, to determine how the steady-state level of GS adenylylation varied when GS is incubated with a highly purified preparation of ATase in the presence of various concentrations of glutamine and a-ketoglutarate and at a number of different mole fractions of Pu. Results of these studies confirmed that cyclic cascades are in fact endowed with all of the properties which were predicted by the theoretical considerations. Verification of these basic principles was obtained also from studies in which the state of adenylylation of GS was measured in permeabilized E. coli cells following their incubation in buffer containing variable concentrations of ATP, UTP, o~-ketoglutarate, glutamine, and Pi .41,42 Application of Molecular Biology Technology It is perhaps worth noting that the discovery and elucidation of the GS cascade as summarized in the above sections was achieved by the application of classical enzymological approaches and technologies. None of the modern techniques of molecular biology or of biochemical genetics was utilized. Indeed, it is questionable whether the application of these latter techniques would have helped or hindered progress in the elucidation of this remarkable cascade system. Nevertheless, as noted earlier, a quantitative study of this bicyclic cascade system was hampered by the instability of the UTase, which precluded its isolation as a homogeneous protein, and also by the fact that, except for GS, the intracellular concentrations of the cascade enzymes are relatively low, making it difficult to obtain sufficient quantities of homogeneous preparations needed for detailed studies of the bicyclic cascade in vitro. To overcome these problems, we resorted to the use of molecular biology approaches to obtain strains ofE. coli which produced 800-, 500-, and 70-fold more UTase, ATase, and PII protein, respectively, than the wild-type strain. 43,44 Using these strains, Rhee and colleagues obtained a substantial quantity of a homogeneous preparation of each one of the cascade enzymes. In a monumental e f f o r t , 45 they determined the values of 21 interaction constants that govern the protein/protein and protein/ 41 U. Mura and E. R. Stadtman, J. Biol. Chem. 256, 13014 (1981). 42 U. Mura, P. B. Chock, and E. R. Stadtman, J. Biol. Chem. 256, 13022 (1981). 43 S. G. Rhee, S. C. Park, and J. H. Koo, Curr. Top. Cell. Regul. 27, 221 (1985). 44 H. S. Son and S. G. Rhee, J. Biol. Chem. 262, 8690 (1987). 45 S. G. Rhee, W. G. Bang, S. C. Park, J. H. Koo, and K. H. Min, in "Dynamics of Soluble and Immobilized Enzymes" (P. B. Chock, L. Tsou, and C. Y. Huang, eds.), p. 128. Springer-Verlag, Amsterdam, 1987.
[60]
DISCOVERY OF GLUTAMINE SYNTHETASE CASCADE
807
effector interactions which are involved in various steps of the bicyclic cascade. Then, in order to simulate in vitro the characteristics of the GS cascade, a series of experiments was carried out in which highly purified preparations of the cascade enzymes and the Pu protein were mixed together in amounts equivalent to those found in crude extracts ofE. coli. In this study, the effects of variations in the relative concentrations of glutamine and a-ketoglutarate were examined under three different conditions, namely: (1) in the presence ofUTP, Pi, ATP, UTase, and PII only (to mimic the operation of the uridylylation/deuridylylation cycle alone), (2) in the presence of ATase, ATP, and varying ratios of Pu and PII" UMP (to mimic operation of the adenylylation/deadenylylation cycle alone), and (3) in the presence of all substrates and cascade enzymes (to mimic the behavior of the coupled bicyclic cascade). Suffice it to say that the results of these studies confirmed in every important detail theoretical predictions of the cascade model, and illustrated directly the remarkable regulatory features of such cascades .45 It was evident from the results of these studies that interconvertible enzyme cascades serve as metabolic integration systems. By means of allosteric and substrate site interactions, the interconvertible enzymes are programmed to sense fluctuations in the concentrations of a multiplicity of metabolites. This leads to automatic adjustments in the specific activities and kinetic constants of the several cascade enzymes. Through this system the multiple inputs are integrated and registered as a single output, the fractional modification of the target enzyme, and thereby determines its specific catalytic activity. Role of UTase and Pu in Regulation of Glutamine Synthetase Formation In the initial studies on the regulation of GS activity, Woolfolk noted that the level of GS in E. coli was dependent on the availability of nitrogen in the culture medium. In the meantime, the mechanism that underlies the nitrogen control of GS synthesis was under extensive investigation in the laboratory of M a g a s a n i k 46'47 and K u s t u . 48'49 In both laboratories, a detailed genetic analysis of the GS cascade led eventually to the demonstration that UTase and the Pn protein are involved in the nitrogen control of GS levels. Such roles were suggested also by the studies of Rhee and 46 B. Magasanik, Annu. Rev. Genet. 16, 135 (1982). 47 B. Magasanik, TIBS 13, 475 (1988). 48 S. Kustu, K. Sei, and J. Keener, in "Regulation of Gene Expression" (I. R. Booth and C. F. Higgins, eds.), p. 139. Cambridge Univ. Press, London, 1986. 49 j. Keener, P. Wong, D. Popham, J. Wallis, and S. Kustu, in " R N A Polymerase and Regulation of Transcription" (W. S. Reznikoff, R. R. Burgess, J. E. Dahlberg, C. A. Gross, M. T. Record, Jr., and M. W. Wickens, eds.), p. 159. Elsevier, New York, 1987.
808
APPENDIX
[60]
j,s ~ m j'
GSactivity
HzO
"~
UMP
\ p]i_U ~~P I _UTd _ ~P,]]
PPi
ADP 10 P. ~ J'
UTP
ATP
~ PPi
HzO (~
Pi
N ~ I ~ N I
GS synthesis
ADP
ATP
FIG. 4. The cyclic cascade of glutamine synthetase (GS) regulation. Interrelationship between the uridylylation cycle, the adenylylation cycle, and the phosphorylation cycle; the reciprocal controls of these interconversions by L-glutamine (Gin) and a-ketoglutarate (a-KG) are shown; 0) indicates stimulation, O indicates inhibition. Abbreviations: GS, glutamine synthetase; PII, regulatory protein; Ata and ATd, adenylylation and deadenylylation sites, respectively, on the bifunctional adenylyltransferase; UTu and UTd, uridylylation and deuridylylation (uridylyl-removing) sites, respectively, on the bifunctional uridylyltransferase. NRI, glnG product also known as NTRC; NRuK and NRap, glnL product (also known as NTRB) catalyzing phosphorylation and dephosphorylation of NR~, respectively.
colleagues, showing that the repression of GS synthesis which occurs when E. coli is grown in a nitrogen-rich medium is regulated in part by the intracellular concentrations of Pzi, UTase, and ATase. 45The participation of Pu and UTase in the repression of GS synthesis was ultimately clarified by the elegant studies of Ninfa and Magasanik 5° and Keener and Kustu. 51 They showed that transcription of the structural gene for GS is under the control of several gene products, two of which, glnG (ntrC) and glnL (ntrB), are members of the gln operon. The product of the glnL gene (NRII) is a protein kinase that catalyzes the phosphorylation of the glnG so A. J. Ninfa and B. Magasanik, Proc. Natl. Acad. Sci. U.S.A. 83, 5909 (1986). 51j. Keener and S. Kustu, Proc. Natl. Acad. Sci. U.S.A. 85, 4976 (1988).
[61]
ELONGATIONFACTORS
809
product (NRI) and thereby converts it to a form, NRI-P, which can activate glnA transcription. However, the cyclic interconversion of the glnG product between phosphorylated (NRI-P) and unphosphorylated (NRI) forms is dependent upon the concentration of the PII protein which stimulates the dephosphorylation of NRI-P. Whether the effect of Pn is to convert the NRu from a kinase to a phosphatase or serves only as an effector which together with NRII accelerates the spontaneous dephosphorylation of NRI-P appears unsettled. In any case, it became evident from these studies and those summarized above that the regulation of GS activity on the one hand and the regulation of GS formation on the other are tightly linked via interconversion of PII between its unmodified and uridylylated forms (Fig. 4). It is in fact the UTase which via allosteric interactions senses changes in the concentrations of a-ketoglutarate and glutamine and thereby dictates the steady-state levels of PII and PII"UMP; these in turn specify the activities of NRII and ATase, which in turn determine the steady-state levels of NRI-P and adenylylated GS, and hence the rate of GS synthesis and GS activity.
[61] D i s c o v e r y , R e s o l u t i o n , P u r i f i c a t i o n , a n d F u n c t i o n o f Elongation Factors By KIVIE MOLDAVE
In the early 1950s, most of the research on protein biosynthesis focused on the cellular components required for the incorporation of amino acids into proteins in cell-free extracts in vitro. Phil Siekevitz ~found that incubation of rat liver mitochondria, cytosol, and microsomes (fragments of endoplasmic reticulum membranes containing ribosomes, nonribosomal proteins, endogenous mRNA, lipids, etc.) accounted for most of the incorporation of amino acids observed when whole homogenates were used; mitochondrial oxidative phosphorylation was necessary. Zamecnik and Keller 2 obtained a more refined system that consisted of microsomes, a nondialyzable heat-labile fraction from the cytosol, and an ATPgenerating system; the energy requirements provided by mitochondrial oxidative phosphorylation were replaced by ATP and an ATP-generating P. S i e k e v i t z , J. Biol. Chem. 195, 549 (1952). 2 p. C. Z a m e c n i k and E. B. Keller, J. Biol. Chem. 2119, 337 (1954).
METHODS IN ENZYMOLOGY, VOL. 182
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