86
Bioehimicn n Biophysiea dc,'a. 1076 (1991) 86-90 © 1991 Elsevier Science Pu[:l/sh~s B,V. tBi,:anedical Division) 016%4838/91/$03.50 ADONIS O16"14838g10uO69D
BBAPRO33~8
Functional consequences of substitution of the active site (phospho)histidine residue of Escherichia colt succinyl-CoA synthetase R a m a n a t h M a j u m d a r t, J o h n R. G u e s t 2 a n d W i l l i a m A. Bridget t PD~partment ~,/Biochemistry, Um~e,,sity o/Alberta, Edmonton, fCanada) and z Deparlme, t o~ Molecular Biology and Biotechaolog¢, Universizy o/SheffleR~ She/field (IX K.) (Received ! April 1990) Key words: SuccinyI-CoA synthel~se: Site-directed mutant: Enzyme mechanism; (E. coli)
SuceinyI-CoA synthetase (EC 6.ZLS, sueclnale:CoA llgase {ADP-fofmlng)} of Escheriehia ¢oli is an az~ z tetramer, with the active site believed to be located at the point of eonlacl between the two subunit types. 1I has been previously established that the reaetinn involves the intermediate parlicipation of a phospholfflated enzyme form in the process of catalysis. The site of plmsphorylalion (His-2416)and the binding sites for the substrates ADP ~JadATP are located in the a subunit, and the suceinate aml CoA binding sites are in/~. A mutant form of this enzyme, with the active site histidine residue replaced by a s t u t e , has been produced in large quantities and purified to homogeneity. This form appears to be indistinguishable from the native enzyme with respect to its subanit assembly, but has no ability t~ e~alyze the overall reaction. As expected, the His-246a--, Asp mutant is incapable of tmdergeing i/Imspharylatian. We ~ave developed an assay based upon the arseoelysis of suecinyI-CoA that effectively isolates lhe partial reaction that occurs in the portion of the active site contributed by tim// subanig this reaction does no~ involve covalent participation of His-246a. We have found that the His-246a ~ Asp mutant is also devoid of activity in this arsenolysis reaction, indicating that an intact His.246a is required for the establishment of the mieroenvimnment in this portion of the active site that is required for the corresponding step of the overall reactinn. Introduction
The metabolic role of succinyl-CoA synthetase is catalysis of the substrate-levei phosphorylation step of the tficarboxylic acid cycle, the synthesis of nucleoside triphosphate coupled to the cleavage of succinyI-CoA. The reaction is believed 1o proceed in three distinct partial reactions involving two discrete phosphorylated intermediates, namely tightly-hound succinyl phosphate (E(Succ-PO3)) and a phosphoenzyme intermediate (Epo~): E + SuccinyI-CoA + Pl ~ CoA + E(Succ-PO~ ) E{Succ-POj) ~ $uceinale +
E-PO~
{1) i2)
]~-PO3 +ADP ~ E + ATiP
(3)
Sum: SuccinyI-CoA + Pi 4- ADP ,~ Succinate 4-CoA +ATP
{4}
Abbreviations: CoA. coergyme A; As i, inorganic arsenate; SDSPAGE sodium do4~c~ylsulphate polyacrylamide gel electrophoresis. Co~¢spendenc¢: W.A. Bfidgcr, Deparlment of Biochemis~, tlniversity of Alberto, Edmonton, Alberta, Canad~ TtG 2H7.
The enzyme from E. colt, the subject of this communication, has an a~fl2 subunit structure (M r for a--29600; Mr for ,8=41400) [1]. Formation of E-PO3 involves the phosphor,/lation of a histidine residue in the a subunit (His-246a) which also contains binding sites for ADP/ATP [2]. Sites for attachment of succinate and CoA are believed to be located on the fl subunit {for further review, see Rcfs. 3 and 41. The a and ~/ suburtits are encoded by the sued and ~ucC genes, respectively, of the E. colt sue operon [5,6]. These genes have been cloned, sequenced and inserted into a high-level expression vector [6,7]. Much of the interest in the mechanism of action of this enzyme has focussed on the catalytic participation of the phosphohistidine residue. By rapid mixing and qaenclzing measurements [g]. it has been established that E-PO3 is an obligatory intermediate that must turn over with each catalytic throughput. When measured in the absence of other substratcs, however, the ADP ATP exchange reflecting E-PO3 formation (Step 3 above) is slow, but this is stimulated to the net catalytic rate by the occupancy of the succinate and CoA binding sites; this phenomenon is known as 'substrate synergism' [8]. The availability of cloned succinyl-CoA
87 symhetase and, in particular, a mutant in which the potential phosphohistidine residue is changed to aspartate [7], have presented new opportunities for investigation of the communication between the various subsites at the active centre of this enzyme. In this paper, we report tha~ the His-246a---, Asp mutant is devoid of detectable catalytic activity for the overall reaction, as expected. Moreover, and perhaps unexpectedly, we zhow that this mutant derivative is incapable of catalysis of partial reaction I (above), a step of the reaction in which the histidine residue doer not participate covalently and which is believed to take place on the other side of the a/[i contact at the active site. Materials and Methods .Materials Succinyl-CoA, potassium arsenate, ADP and ATP were purchased from Sigma~ Restriction enzymes and T4 DNA ligase were obtained from Bethesda Research Laboratories. [¥-32PIATP (4500 Ci/mmol) was obtained from New England Nuclear. Bacterial strains and plasmids E. coli strain TK3D18 (z~lkdp-suc]DlS,/l[gal-bio]) is a null strain in which the sucC and sued genes are deleted [9]; this was kindly provided by Dr. Wolfgang Epstein of the University of Chicago. pGS202 is a pJLAS03 derivative in which the sucCD genes are expressed from tandem k promoters, and pGS201 is a pBR3.22-derivative in which the sucCD genes are expressed from the ter promoter and the sucC gene encodes a His-246a --* Asp substitution [7]. Plasmid construction The appropriate Clal-BamH1 fragment containing the CAC(His) ---,GAC(Asp) mutation in sued was cut from pGS'201 [7] and this was used to repla~ the corresportding fragment in pGS202 to construct a new plasmid pMB204 for the overproduction of the mutant form of succinyl-CoA synthetase (see Fig. 1). pMB204 is thus ultimately derived from pJLA503 and is similar to pGS20"2 except for the single eodon replacement, His-246a --. Asp in the DNA insert. TK3D18 cells (the null strain for succinyI-CoA symhetase) were separately transformed with pGS202 and pMB2O4 according to standard methods [11]. Production of normal and mutant succinyl-CoA synthetase Overnight cultures of pGS202o or pMB204-transformed TK3Dll] cells were grown on a modified LB medium [11] containing KCI in place of NaCI. These were used to inoculate 10 ! fermenter cultures which were grown with full aeration for 3 h at 28°C. The temperature was the shifted to 42°C and the cells were harvested at stationary phase. Cell extracts were pre-
~ A ~ s 8 5 7
Fig. t. Map of pMB204. Ire vector tar overespressionof mulant succinyl-CoAsynthetase.The elementsshownincludetandempromoters from phage 3,, the lemperature-sensitive~. clts promoter,the fd-transcription terminator(tfd), a ribasgrnebinding scquen~ (xbs) and the sueC and sued geneseneodlngthe/~ and a subunitsof E. cull succinyI.CoAsymhetase.A CAC(His).-*GAC(Aspl mutation is introducedinto the sued gene(seete~t). pared by sonic disruption 1121. Purification of both the normal and mutant varieties of the enzyme followed established protocols [1]; the inactive mutant enzyme wa~; detected throughout the purification process by means of immunological probes and SDS-PAGE (see below). Enzyme assay SuccinybCoA synthetase activity was routinely measured with the standard spcctrophotometric assay t13] that is based upon the increase in absorbance at 232 nm accompanying formation of succinyl-CoA. For measurement of the arsenolysis of succinyl-CoA (see below), the eaetion mixture contained 50 mM Tris-HCi (pH 7.4), 50 mM KCI, 30 I~M succinyI-CoA, 1 mM potassium arsenate, 10 mM MgCI2, and 0.1-0.4 units of enzyme in a total vol. of I ml at 37°C. The arsenolysis of suecinyI-CoA was measured by the decrease in absorbance at 232 nm. SDS-PA GE and immuneblotting SDS-PAGE was done according to Laemmli [141 using separating gels made up with 10,~ aerylamide. Immunobiot analysis was performed after electrophoretic transfer to nitrocellulose membranes. The membranes were blocked by treatment with a 5% (w/v) solution of non-fat dried milk in Tris-buffered saline fiBS) for 2 h at room temperature. The membranes were then incubated overnight with antiserum to £. eoli suecinyl-CoA synthetase diluted in TBS. After washing twice with TBS containing 0.05% NP-40, membranes were treated with horse radish peroxidase-conjugated goat anti-rabbit lgG diluted 2000-fold in 35~ (w/v) bovine serum albumin for 3 h at 37°C. Membranes were then washed twice in with 0.05% NP-40 in TB$
g8
A
and colouf was developed by the addition of 4-chlorol-naphthoi substrate in the presence of 0.015% H202.
B
Phosphorylatwn o] enzyme Using methods previously described [15], the purified proteins were subjected to phosphorylation by [Y32P]ATP and the protein was separated from unreacted nueleotide. After SDS-PAGE the labelled protein was located either by autoradiography or by scintillation coan'~ing of solnbilized slices [2[ For antoradiography, gels were dried onto Wluttman No. 3 filter paper and were exposed to Kodak X-Omat AR film at - 7 0 ° C .
~J=~
•
~-
7..
P~ a - 4 ~
Results and D i s c u ~ o n
Overexpres~ion and purification o/mutant succinyI-CoA synthetase Buck and Guest [7l have demonstrated the utility of the pJLAS03 system for the overexpressioa of wild-type suceinyl-COA synthelase; under optical conditions cells transformed with the sucCD-contaimng pGS202 (derived from lxILA503) produce this enzyme as about 10% of cellular protein. However, these workers found that cells transformed with pGS201 (a pBR322-derivative which encodes the His-246,~---+Asp substitution and expresses the sueCD genes from the tes promoter) produce no detectable activity, but possible effects of the mutation on polypeptide folding or susceptibility to proteolysis were not excluded. Using the expression vector pMB204, however, we find that massive m o u n t s of the mutant protein may be produced (see Fig. 2A). The identity of the over-expressed subunits was confirmed by immunoblotting (Fig. 2B). Purification of the mutant form of the enzyme required no modification to the methods that we routinely use for the preparation of wild type E. coli suceinyl-CoA synthetase. Most significantly, the purified mutant proteL1 emerged as a single symmetrical peak from gel filtration columns, indistinguishable in its behaviour from the native E. coli enzyme where the latter has been established to be an a2B2 tetramer [1], and very clearly resolved from the pig hcert enzyme which is known to be an a~ dimer [1]. The His-246,~ ~ Asp mutation thus appears to be silent with respect to protein stability, general folding properties ~nd assembly of the tetramcr. We cannot exclude the possibility, of course, that the substitution causes small but important changes in the folding of the polypeptide chain in the vicinity of the active site. But in consideration of the apparently native conformation of the mutant protein, it may be significant that the His-246~, --* Asp substitution introduces a negative charge at position 246n, and it is known that refolding of the native enzyme from the separated subunits is greatly facilitated by the binding of Pi or by phosphorylation of His-246~x [16], processes that introduce negative charge at the equivalent place in the protein.
1
2
3
4
1
2
3
4
Fig. 2. {A, left) SDS-PAGEof crude extracts and pnrifiedsuccinyl.. CoA synthetas~: ultrasonicexlre~'tswere prepared after thermoinduction of pgS202 and pM~04 transformantsof TK3DIS cells~ Details are given under Materials and Methods. Lane t, 40 ~g punfied E. eel[ SCS: lane 2, 2(]0pg erode e~tract of TK3D]$ cells; lane 3, 150 p.gof crude extractof TK.3DI8 cells(pGS202);and lane4, 200 ItS crude extract of TK3DI8 cells (pMB204). Gels are ~lain¢~:l with Coomassieblue.(13,right)Immunoblotanalysisof trade exlracts and purifiedSCS: a cornpar~on~e! to thai shown in Panel A was electroblottedto nitrceellulo~and probedwith antiserumto purified E. cMi succinyl-CoAsynthe*.aseas de~ribcA under Materials and Methods. P~lein samplesin each lane ate as th:~rib~l for Panel A,
Lack of activity of the mutant enzyme When measured with the normal assay procedure that is based upon the overall synthesis of succinyl-CoA (reaction 4 above), no activity could be detected with the purified mutant protein. This is in keeping with the observations of Buck and Guest [7], who found no increase in enzyme activity in extracta of cells transformed by pGS201, and with the role proposed [8] for transient phosphorylation of His-246a as an obligatory step in the catalytic process (see reactions 2 and 3). As much as 0.3 mg of mutant protein was tested with our standard assay without delectable production of produel over 20 rain. Under these conditions we would have ~eadily detected activity that was equivalent to four orders of magnitude less than that of the native enzyme. Although the His-246a --* Asp mutant was found to have no demonstrable ability to catalyze the overall reaction, it remained a possibility that it might still be able to undergo phosphorylation at the substituted residue. Intermediate phosphorylation of aspartate residues has been found to occur in the action of other enzymes - a prominent example is Na+/K+-ATPase [17]. To test this, both the native and the His-246a ---, Asp mutant enzyme were treated with [y-32P]ATP, fol-
89 OL
+
+
pMltZ04.-~
i
1COC~.
- + -
81000,
£
|
6000.
4000,
pMnO04 (mtttant)
•
~k_:_..~.= :... 5
10
15
20
25
30
Slice Number Fig. 3. Incorporalion of ~P into native ar,d mutant suecinyl-CoA synthetase. Both nzdve and mutant ~uccinyl-~:,Agynthet~+.meWere incubated with ['p~PIATP as described in Materials and Methods. After SDS-PAGE, gels were sU+ed ~ d counted for radioactivity (bottom) or ttsed for aatoradiography(top). The mabilltiesof a and/] subunitstandardsa~u8i.vCn,
lowed by. SDS-PAGE. The results (Fig, 3) show that the mutant enzyme incorporates no detectable 32p under conditions where native succinyl-CoA synthetase is fully phosphorylated at His-246a. This therefore suggests that Asp-246a in the mutant enzyme is not in the appropriate orientation to serve as an accepter of the phospho~l group of ATP. In view of the known involvement of transient phosphorylation of His-246a in catalysis by suecinyl-CoA syathetase, the inability of the enzyme to catalyze the overall reaction (Step 4) or phosphoryl transfer (Step 3) c a m e as no surprise. However, especially in view of the fact that the binding sob-sites for saccinyI-CoA are thought to be located on the part of the active site contributed by the neighbottring ~ subunit [2-4], we wished to determine the ability of the mutant enzyme to catalyze a step of the reaction in which His-246a has no covalent participation. We believed that Step 1, the phosphorolysis of succinyI-CoA by Pi, could be effeclively isolated and measured by the substitmion of arsenate (Asi) for Pi, t~king advantage of the known instability of nfixed anhydrides of arsenate [18]. The measurement of rate of the arsenolysis reaction E+ Succinyl-CoA.+As .~ E(Suec-As03)~ E+ Soccinate÷Ast ('oA
H~D
would thus be an effective probe of the ability of the mutant enzyme to catalyze the equivalent phosphorolysis ~tep. The native enzyme was found to be an effective catalyst for the arsenolysis reaction (see Materials and Methods for conditions of assay). The specific activity for ar.~nolysis was found to be = 3.0 p m o l - r a i n - l . mg -+, about one-tenth of the specific activity for the standard assay of the overall reaction. Table I shows the Vm~ and Km values for succinyl-CoA and arsenate, Also shown in Table I are the kinetic parameters determined in the presence of A D P - this addition was found to have no detectable effect on the arsenolysis reaction. This is significant because it has been shown [8] that the rate of Step 3 (i.e., the A D P ~ ATP exchange) is markedly ~imulated by succinyl.CoA bind. ing, but the present result indicates tl~tt a reciprocal kind of substrate synergism (promotion of Step 1 by A D P attachment) does not occur. Also in Table I, we indicate that the arsenolysis reaction is undetcctable w h e n native suecinyl-CoA synthetase is replaced by the His-246a ~ Asp ~mtant enzyme+ With as much as 0 J mg of mutant protein added to the arsenolysis mixture, no activity could be measured. This shows that the His-246a --, Asp substitution affects no only the steps of reaction that oect~r on the a subunit's contribution to the active site ti',at involve phosphoryl transfer to and from the histidine residue, but also that the effects of the substitution must extend to the microenvironment across the subunit contact to the region of the active site where the cleavage of succiny|-CoA takes place. Although all of our data and TABLE [ lOaeZic properties of nati~e and Hgs-246a ~ Asp ran:ant succi~yI-Coa symkezares for catalysis of t~e ar~.nolysis of succinyl.CoA Kinetic parameters were determined from linear double ~ciprecal plots. Co,ditions for ashy are as described in Materials and Moth. ods~ Whe~ [succinyI.CoA]was varied, arsenate was fixed at 1 raM; when [atsenatel was varied, stw.cinyl-CoAwas fixed at 25 FtM, Fog measurementswith native enzyme, 3 ttg/ml of protclr, was i~..cluded. Mutant enzymewas tested in concentrationsranging to 0.1 mg/ml. Substrale
Nativeenzyme succinyl-CoA suecinyI.CoA
ADP (0.12 raM)
Km (p.M)
+
Vrm~ (pmo],min -I .rag - I )
7.6 7.7
3.0 3.0
arsenate
-
26,9
3.0
arsenale
+
27,0
3.0
-
0.C0" o.00 •
His-246o--+Asp mutant succinyl-CoA +/ armnatu +/ -
-
• No activity ,~vass , ~ ~ up to 0.l tag of mutant enzymein the assay mixture.TEe assay systemwould have readilydetected activ. ity of ;~0.003 pmol-min-Lmg-t.
90 observations indicate that the mutant enzyme is folded and assembled like the native cbunterpart, it thus appears to he devoid of any vestige of catalytic activity. This work temple,'- nts other studies of modification of residue, ,, subunit. Chemical modification of eysteine sulfhydryl groups by a coenzyme A affinity analogue [19] suggested that Cys-325B is accessible to the CoA binding site in the 18 subunit. Recent site-directed mutation of Cys-325B to a glycine residue produced a variant that retains nearly full catalytic activity but has lost its susceptibility to inactivation by the CoA analogue [20], indicating that this residue near the Cterminus of/~ is a non-essential active site residue. In another recent Frobe of function of tryptophan residues in the ,a subunit, Nishimura and his colleagues [21] have found that replacement of Trp-//43 or Trp-//248 by phenylalanine residues produces a fully active enzyme, but that the fluorescence of these tryptophan residues is responsive to binding of CoA. The various mutagenesis and modification studies are ~hus presenting an evolving but still vague picture of the structural organization of the active site at the a/~ contact region. Completion of the structure determination by X-ray crystallography, presently underway in this laboratory [221, is thus highly desirable.
References
Acknowledgements
W.A. 0983) J. Bid. Chem.25S, 14116-14119. 16 Wolodko,W:I'. and Bridget, W.A.(1987) Biochem.Cell Biol. 65,
We thank Mr. IEd Brownie for his expert assistance with the production and purification of the native and mutant enzymes. ILM. is a Fellow of the mOert.a Heritage Foundation for Medical Research. This work was supported by a grant (MT-2805) frem the Medical Research Council of Canada.
1 Wolodko,W.T., Kay, C.M.and Br/dgeLW.A.(1986)Bica:hemls|ry 25. 5420---5425. 2 Pearson, P.H. and BrMger. W.A. {]975) J. Biol, Chem. 250. 8524-8~29,
3 Bridger, W.A, {1974) in ]'he EnzymesVol, X (Buyer, P,D.. ed.), pp. 581~606, Academic Press. New YorL 4 Nishimara, J.S. (1986) in Advancesin Eazymol~gyand Related Areas of Mol~ular Biology'4ol, 58 {Meistcr,A.. e~.Lpp. 141-172, John Wiley, New York. 5 BucU D., Spencer,M.E. az~dGull. J.R. (19E6)J. Gun. Mierobiol. 132, 1753-1762. 6 Buck, D., Spencer, M~E.and Guest. J.R. (1985) Biochemistry24, 6245-6252. 7 Buck, D- and Guest,J.R. (1989) Binchem.J~ 260, "/37 747. 8 Bridget'.W.A.. Millan. W.A. and Buyer,P.D. (1968) Biochemis*.ry 7. 3608-3616. 9 Rhoads, D.B., Laimins. L. and Epstein, W. (1978) J~ Bacteriol. 135. 445-452. l0 Schauder, B.~ BlOcker, H.. Frank. IL and McCarthy, J.E.G. (!087) Gone 52. 279-283.
t|. Manlatls, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Maau,'d. Cold SpringHarbor Laboratory, Cold Spring Harbor.New York. 12 Wolodko, W.T.. BroweJe. E.R. and Bridget, W.A. (t980) J~ B~clcriol. 143, 23l-2~7. 13 Bridget, W.A., Ramale7, R.F. and Buyer, P.D. (t969) in MetV.oas ia Enzymo]ogyVol. X111 (Loweaslein. J_M.. ed.), pp. 70-75, Academic Press, New York. 14 LaemmlL U.K. (1970) Nature 227, 680-685, I5 Wolodko. W.T., Bmwnie~ E.IL. O'ConnoL M.D. and Bridger.
452-45"/.
17 Nishigagi, 1., Chert, F.T. and Hokirt. I..E. 0974) J. BioL Chem. 249. 491t -4916. 18 Hurling,J. and Vdick, S.F. (19M) J. Biol.Chem.207, 867-878. 19 Collier, G.E. and Nishimura. J.S~ (197g) J. Biol. Chum. 2.$3. 4938-4943. 20 Mann~ CI., Hurdles, S.C. and. Nishimora, J.S. (1989) J. BioL Chem. 264, 14Y1-1460. 21 Nishimura, J.S.,Mann, CJ., Yi~arra.J.,Mitchell,T. and Horowitz, P.M. (1990} Biochemistry29, 862-865.
22 Wolodko, W.T.. James. M.N.G. and Bridger, W.A. {1984)J. Biol. Chem. 259. 5316-:~320.
Bioehimicn n Biophysiea dc,'a. 1076 (1991) 86-90 © 1991 Elsevier Science Pu[:l/sh~s B,V. tBi,:anedical Division) 016%4838/91/$03.50 ADONIS O16"14838g10uO69D
BBAPRO33~8
Functional consequences of substitution of the active site (phospho)histidine residue of Escherichia colt succinyl-CoA synthetase R a m a n a t h M a j u m d a r t, J o h n R. G u e s t 2 a n d W i l l i a m A. Bridget t PD~partment ~,/Biochemistry, Um~e,,sity o/Alberta, Edmonton, fCanada) and z Deparlme, t o~ Molecular Biology and Biotechaolog¢, Universizy o/SheffleR~ She/field (IX K.) (Received ! April 1990) Key words: SuccinyI-CoA synthel~se: Site-directed mutant: Enzyme mechanism; (E. coli)
SuceinyI-CoA synthetase (EC 6.ZLS, sueclnale:CoA llgase {ADP-fofmlng)} of Escheriehia ¢oli is an az~ z tetramer, with the active site believed to be located at the point of eonlacl between the two subunit types. 1I has been previously established that the reaetinn involves the intermediate parlicipation of a phospholfflated enzyme form in the process of catalysis. The site of plmsphorylalion (His-2416)and the binding sites for the substrates ADP ~JadATP are located in the a subunit, and the suceinate aml CoA binding sites are in/~. A mutant form of this enzyme, with the active site histidine residue replaced by a s t u t e , has been produced in large quantities and purified to homogeneity. This form appears to be indistinguishable from the native enzyme with respect to its subanit assembly, but has no ability t~ e~alyze the overall reaction. As expected, the His-246a--, Asp mutant is incapable of tmdergeing i/Imspharylatian. We ~ave developed an assay based upon the arseoelysis of suecinyI-CoA that effectively isolates lhe partial reaction that occurs in the portion of the active site contributed by tim// subanig this reaction does no~ involve covalent participation of His-246a. We have found that the His-246a ~ Asp mutant is also devoid of activity in this arsenolysis reaction, indicating that an intact His.246a is required for the establishment of the mieroenvimnment in this portion of the active site that is required for the corresponding step of the overall reactinn. Introduction
The metabolic role of succinyl-CoA synthetase is catalysis of the substrate-levei phosphorylation step of the tficarboxylic acid cycle, the synthesis of nucleoside triphosphate coupled to the cleavage of succinyI-CoA. The reaction is believed 1o proceed in three distinct partial reactions involving two discrete phosphorylated intermediates, namely tightly-hound succinyl phosphate (E(Succ-PO3)) and a phosphoenzyme intermediate (Epo~): E + SuccinyI-CoA + Pl ~ CoA + E(Succ-PO~ ) E{Succ-POj) ~ $uceinale +
E-PO~
{1) i2)
]~-PO3 +ADP ~ E + ATiP
(3)
Sum: SuccinyI-CoA + Pi 4- ADP ,~ Succinate 4-CoA +ATP
{4}
Abbreviations: CoA. coergyme A; As i, inorganic arsenate; SDSPAGE sodium do4~c~ylsulphate polyacrylamide gel electrophoresis. Co~¢spendenc¢: W.A. Bfidgcr, Deparlment of Biochemis~, tlniversity of Alberto, Edmonton, Alberta, Canad~ TtG 2H7.
The enzyme from E. colt, the subject of this communication, has an a~fl2 subunit structure (M r for a--29600; Mr for ,8=41400) [1]. Formation of E-PO3 involves the phosphor,/lation of a histidine residue in the a subunit (His-246a) which also contains binding sites for ADP/ATP [2]. Sites for attachment of succinate and CoA are believed to be located on the fl subunit {for further review, see Rcfs. 3 and 41. The a and ~/ suburtits are encoded by the sued and ~ucC genes, respectively, of the E. colt sue operon [5,6]. These genes have been cloned, sequenced and inserted into a high-level expression vector [6,7]. Much of the interest in the mechanism of action of this enzyme has focussed on the catalytic participation of the phosphohistidine residue. By rapid mixing and qaenclzing measurements [g]. it has been established that E-PO3 is an obligatory intermediate that must turn over with each catalytic throughput. When measured in the absence of other substratcs, however, the ADP ATP exchange reflecting E-PO3 formation (Step 3 above) is slow, but this is stimulated to the net catalytic rate by the occupancy of the succinate and CoA binding sites; this phenomenon is known as 'substrate synergism' [8]. The availability of cloned succinyl-CoA
87 symhetase and, in particular, a mutant in which the potential phosphohistidine residue is changed to aspartate [7], have presented new opportunities for investigation of the communication between the various subsites at the active centre of this enzyme. In this paper, we report tha~ the His-246a---, Asp mutant is devoid of detectable catalytic activity for the overall reaction, as expected. Moreover, and perhaps unexpectedly, we zhow that this mutant derivative is incapable of catalysis of partial reaction I (above), a step of the reaction in which the histidine residue doer not participate covalently and which is believed to take place on the other side of the a/[i contact at the active site. Materials and Methods .Materials Succinyl-CoA, potassium arsenate, ADP and ATP were purchased from Sigma~ Restriction enzymes and T4 DNA ligase were obtained from Bethesda Research Laboratories. [¥-32PIATP (4500 Ci/mmol) was obtained from New England Nuclear. Bacterial strains and plasmids E. coli strain TK3D18 (z~lkdp-suc]DlS,/l[gal-bio]) is a null strain in which the sucC and sued genes are deleted [9]; this was kindly provided by Dr. Wolfgang Epstein of the University of Chicago. pGS202 is a pJLAS03 derivative in which the sucCD genes are expressed from tandem k promoters, and pGS201 is a pBR3.22-derivative in which the sucCD genes are expressed from the ter promoter and the sucC gene encodes a His-246a --* Asp substitution [7]. Plasmid construction The appropriate Clal-BamH1 fragment containing the CAC(His) ---,GAC(Asp) mutation in sued was cut from pGS'201 [7] and this was used to repla~ the corresportding fragment in pGS202 to construct a new plasmid pMB204 for the overproduction of the mutant form of succinyl-CoA synthetase (see Fig. 1). pMB204 is thus ultimately derived from pJLA503 and is similar to pGS20"2 except for the single eodon replacement, His-246a --. Asp in the DNA insert. TK3D18 cells (the null strain for succinyI-CoA symhetase) were separately transformed with pGS202 and pMB2O4 according to standard methods [11]. Production of normal and mutant succinyl-CoA synthetase Overnight cultures of pGS202o or pMB204-transformed TK3Dll] cells were grown on a modified LB medium [11] containing KCI in place of NaCI. These were used to inoculate 10 ! fermenter cultures which were grown with full aeration for 3 h at 28°C. The temperature was the shifted to 42°C and the cells were harvested at stationary phase. Cell extracts were pre-
~ A ~ s 8 5 7
Fig. t. Map of pMB204. Ire vector tar overespressionof mulant succinyl-CoAsynthetase.The elementsshownincludetandempromoters from phage 3,, the lemperature-sensitive~. clts promoter,the fd-transcription terminator(tfd), a ribasgrnebinding scquen~ (xbs) and the sueC and sued geneseneodlngthe/~ and a subunitsof E. cull succinyI.CoAsymhetase.A CAC(His).-*GAC(Aspl mutation is introducedinto the sued gene(seete~t). pared by sonic disruption 1121. Purification of both the normal and mutant varieties of the enzyme followed established protocols [1]; the inactive mutant enzyme wa~; detected throughout the purification process by means of immunological probes and SDS-PAGE (see below). Enzyme assay SuccinybCoA synthetase activity was routinely measured with the standard spcctrophotometric assay t13] that is based upon the increase in absorbance at 232 nm accompanying formation of succinyl-CoA. For measurement of the arsenolysis of succinyl-CoA (see below), the eaetion mixture contained 50 mM Tris-HCi (pH 7.4), 50 mM KCI, 30 I~M succinyI-CoA, 1 mM potassium arsenate, 10 mM MgCI2, and 0.1-0.4 units of enzyme in a total vol. of I ml at 37°C. The arsenolysis of suecinyI-CoA was measured by the decrease in absorbance at 232 nm. SDS-PA GE and immuneblotting SDS-PAGE was done according to Laemmli [141 using separating gels made up with 10,~ aerylamide. Immunobiot analysis was performed after electrophoretic transfer to nitrocellulose membranes. The membranes were blocked by treatment with a 5% (w/v) solution of non-fat dried milk in Tris-buffered saline fiBS) for 2 h at room temperature. The membranes were then incubated overnight with antiserum to £. eoli suecinyl-CoA synthetase diluted in TBS. After washing twice with TBS containing 0.05% NP-40, membranes were treated with horse radish peroxidase-conjugated goat anti-rabbit lgG diluted 2000-fold in 35~ (w/v) bovine serum albumin for 3 h at 37°C. Membranes were then washed twice in with 0.05% NP-40 in TB$
g8
A
and colouf was developed by the addition of 4-chlorol-naphthoi substrate in the presence of 0.015% H202.
B
Phosphorylatwn o] enzyme Using methods previously described [15], the purified proteins were subjected to phosphorylation by [Y32P]ATP and the protein was separated from unreacted nueleotide. After SDS-PAGE the labelled protein was located either by autoradiography or by scintillation coan'~ing of solnbilized slices [2[ For antoradiography, gels were dried onto Wluttman No. 3 filter paper and were exposed to Kodak X-Omat AR film at - 7 0 ° C .
~J=~
•
~-
7..
P~ a - 4 ~
Results and D i s c u ~ o n
Overexpres~ion and purification o/mutant succinyI-CoA synthetase Buck and Guest [7l have demonstrated the utility of the pJLAS03 system for the overexpressioa of wild-type suceinyl-COA synthelase; under optical conditions cells transformed with the sucCD-contaimng pGS202 (derived from lxILA503) produce this enzyme as about 10% of cellular protein. However, these workers found that cells transformed with pGS201 (a pBR322-derivative which encodes the His-246,~---+Asp substitution and expresses the sueCD genes from the tes promoter) produce no detectable activity, but possible effects of the mutation on polypeptide folding or susceptibility to proteolysis were not excluded. Using the expression vector pMB204, however, we find that massive m o u n t s of the mutant protein may be produced (see Fig. 2A). The identity of the over-expressed subunits was confirmed by immunoblotting (Fig. 2B). Purification of the mutant form of the enzyme required no modification to the methods that we routinely use for the preparation of wild type E. coli suceinyl-CoA synthetase. Most significantly, the purified mutant proteL1 emerged as a single symmetrical peak from gel filtration columns, indistinguishable in its behaviour from the native E. coli enzyme where the latter has been established to be an a2B2 tetramer [1], and very clearly resolved from the pig hcert enzyme which is known to be an a~ dimer [1]. The His-246,~ ~ Asp mutation thus appears to be silent with respect to protein stability, general folding properties ~nd assembly of the tetramcr. We cannot exclude the possibility, of course, that the substitution causes small but important changes in the folding of the polypeptide chain in the vicinity of the active site. But in consideration of the apparently native conformation of the mutant protein, it may be significant that the His-246~, --* Asp substitution introduces a negative charge at position 246n, and it is known that refolding of the native enzyme from the separated subunits is greatly facilitated by the binding of Pi or by phosphorylation of His-246~x [16], processes that introduce negative charge at the equivalent place in the protein.
1
2
3
4
1
2
3
4
Fig. 2. {A, left) SDS-PAGEof crude extracts and pnrifiedsuccinyl.. CoA synthetas~: ultrasonicexlre~'tswere prepared after thermoinduction of pgS202 and pM~04 transformantsof TK3DIS cells~ Details are given under Materials and Methods. Lane t, 40 ~g punfied E. eel[ SCS: lane 2, 2(]0pg erode e~tract of TK3D]$ cells; lane 3, 150 p.gof crude extractof TK.3DI8 cells(pGS202);and lane4, 200 ItS crude extract of TK3DI8 cells (pMB204). Gels are ~lain¢~:l with Coomassieblue.(13,right)Immunoblotanalysisof trade exlracts and purifiedSCS: a cornpar~on~e! to thai shown in Panel A was electroblottedto nitrceellulo~and probedwith antiserumto purified E. cMi succinyl-CoAsynthe*.aseas de~ribcA under Materials and Methods. P~lein samplesin each lane ate as th:~rib~l for Panel A,
Lack of activity of the mutant enzyme When measured with the normal assay procedure that is based upon the overall synthesis of succinyl-CoA (reaction 4 above), no activity could be detected with the purified mutant protein. This is in keeping with the observations of Buck and Guest [7], who found no increase in enzyme activity in extracta of cells transformed by pGS201, and with the role proposed [8] for transient phosphorylation of His-246a as an obligatory step in the catalytic process (see reactions 2 and 3). As much as 0.3 mg of mutant protein was tested with our standard assay without delectable production of produel over 20 rain. Under these conditions we would have ~eadily detected activity that was equivalent to four orders of magnitude less than that of the native enzyme. Although the His-246a --* Asp mutant was found to have no demonstrable ability to catalyze the overall reaction, it remained a possibility that it might still be able to undergo phosphorylation at the substituted residue. Intermediate phosphorylation of aspartate residues has been found to occur in the action of other enzymes - a prominent example is Na+/K+-ATPase [17]. To test this, both the native and the His-246a ---, Asp mutant enzyme were treated with [y-32P]ATP, fol-
89 OL
+
+
pMltZ04.-~
i
1COC~.
- + -
81000,
£
|
6000.
4000,
pMnO04 (mtttant)
•
~k_:_..~.= :... 5
10
15
20
25
30
Slice Number Fig. 3. Incorporalion of ~P into native ar,d mutant suecinyl-CoA synthetase. Both nzdve and mutant ~uccinyl-~:,Agynthet~+.meWere incubated with ['p~PIATP as described in Materials and Methods. After SDS-PAGE, gels were sU+ed ~ d counted for radioactivity (bottom) or ttsed for aatoradiography(top). The mabilltiesof a and/] subunitstandardsa~u8i.vCn,
lowed by. SDS-PAGE. The results (Fig, 3) show that the mutant enzyme incorporates no detectable 32p under conditions where native succinyl-CoA synthetase is fully phosphorylated at His-246a. This therefore suggests that Asp-246a in the mutant enzyme is not in the appropriate orientation to serve as an accepter of the phospho~l group of ATP. In view of the known involvement of transient phosphorylation of His-246a in catalysis by suecinyl-CoA syathetase, the inability of the enzyme to catalyze the overall reaction (Step 4) or phosphoryl transfer (Step 3) c a m e as no surprise. However, especially in view of the fact that the binding sob-sites for saccinyI-CoA are thought to be located on the part of the active site contributed by the neighbottring ~ subunit [2-4], we wished to determine the ability of the mutant enzyme to catalyze a step of the reaction in which His-246a has no covalent participation. We believed that Step 1, the phosphorolysis of succinyI-CoA by Pi, could be effeclively isolated and measured by the substitmion of arsenate (Asi) for Pi, t~king advantage of the known instability of nfixed anhydrides of arsenate [18]. The measurement of rate of the arsenolysis reaction E+ Succinyl-CoA.+As .~ E(Suec-As03)~ E+ Soccinate÷Ast ('oA
H~D
would thus be an effective probe of the ability of the mutant enzyme to catalyze the equivalent phosphorolysis ~tep. The native enzyme was found to be an effective catalyst for the arsenolysis reaction (see Materials and Methods for conditions of assay). The specific activity for ar.~nolysis was found to be = 3.0 p m o l - r a i n - l . mg -+, about one-tenth of the specific activity for the standard assay of the overall reaction. Table I shows the Vm~ and Km values for succinyl-CoA and arsenate, Also shown in Table I are the kinetic parameters determined in the presence of A D P - this addition was found to have no detectable effect on the arsenolysis reaction. This is significant because it has been shown [8] that the rate of Step 3 (i.e., the A D P ~ ATP exchange) is markedly ~imulated by succinyl.CoA bind. ing, but the present result indicates tl~tt a reciprocal kind of substrate synergism (promotion of Step 1 by A D P attachment) does not occur. Also in Table I, we indicate that the arsenolysis reaction is undetcctable w h e n native suecinyl-CoA synthetase is replaced by the His-246a ~ Asp ~mtant enzyme+ With as much as 0 J mg of mutant protein added to the arsenolysis mixture, no activity could be measured. This shows that the His-246a --, Asp substitution affects no only the steps of reaction that oect~r on the a subunit's contribution to the active site ti',at involve phosphoryl transfer to and from the histidine residue, but also that the effects of the substitution must extend to the microenvironment across the subunit contact to the region of the active site where the cleavage of succiny|-CoA takes place. Although all of our data and TABLE [ lOaeZic properties of nati~e and Hgs-246a ~ Asp ran:ant succi~yI-Coa symkezares for catalysis of t~e ar~.nolysis of succinyl.CoA Kinetic parameters were determined from linear double ~ciprecal plots. Co,ditions for ashy are as described in Materials and Moth. ods~ Whe~ [succinyI.CoA]was varied, arsenate was fixed at 1 raM; when [atsenatel was varied, stw.cinyl-CoAwas fixed at 25 FtM, Fog measurementswith native enzyme, 3 ttg/ml of protclr, was i~..cluded. Mutant enzymewas tested in concentrationsranging to 0.1 mg/ml. Substrale
Nativeenzyme succinyl-CoA suecinyI.CoA
ADP (0.12 raM)
Km (p.M)
+
Vrm~ (pmo],min -I .rag - I )
7.6 7.7
3.0 3.0
arsenate
-
26,9
3.0
arsenale
+
27,0
3.0
-
0.C0" o.00 •
His-246o--+Asp mutant succinyl-CoA +/ armnatu +/ -
-
• No activity ,~vass , ~ ~ up to 0.l tag of mutant enzymein the assay mixture.TEe assay systemwould have readilydetected activ. ity of ;~0.003 pmol-min-Lmg-t.
90 observations indicate that the mutant enzyme is folded and assembled like the native cbunterpart, it thus appears to he devoid of any vestige of catalytic activity. This work temple,'- nts other studies of modification of residue, ,, subunit. Chemical modification of eysteine sulfhydryl groups by a coenzyme A affinity analogue [19] suggested that Cys-325B is accessible to the CoA binding site in the 18 subunit. Recent site-directed mutation of Cys-325B to a glycine residue produced a variant that retains nearly full catalytic activity but has lost its susceptibility to inactivation by the CoA analogue [20], indicating that this residue near the Cterminus of/~ is a non-essential active site residue. In another recent Frobe of function of tryptophan residues in the ,a subunit, Nishimura and his colleagues [21] have found that replacement of Trp-//43 or Trp-//248 by phenylalanine residues produces a fully active enzyme, but that the fluorescence of these tryptophan residues is responsive to binding of CoA. The various mutagenesis and modification studies are ~hus presenting an evolving but still vague picture of the structural organization of the active site at the a/~ contact region. Completion of the structure determination by X-ray crystallography, presently underway in this laboratory [221, is thus highly desirable.
References
Acknowledgements
W.A. 0983) J. Bid. Chem.25S, 14116-14119. 16 Wolodko,W:I'. and Bridget, W.A.(1987) Biochem.Cell Biol. 65,
We thank Mr. IEd Brownie for his expert assistance with the production and purification of the native and mutant enzymes. ILM. is a Fellow of the mOert.a Heritage Foundation for Medical Research. This work was supported by a grant (MT-2805) frem the Medical Research Council of Canada.
1 Wolodko,W.T., Kay, C.M.and Br/dgeLW.A.(1986)Bica:hemls|ry 25. 5420---5425. 2 Pearson, P.H. and BrMger. W.A. {]975) J. Biol, Chem. 250. 8524-8~29,
3 Bridger, W.A, {1974) in ]'he EnzymesVol, X (Buyer, P,D.. ed.), pp. 581~606, Academic Press. New YorL 4 Nishimara, J.S. (1986) in Advancesin Eazymol~gyand Related Areas of Mol~ular Biology'4ol, 58 {Meistcr,A.. e~.Lpp. 141-172, John Wiley, New York. 5 BucU D., Spencer,M.E. az~dGull. J.R. (19E6)J. Gun. Mierobiol. 132, 1753-1762. 6 Buck, D., Spencer, M~E.and Guest. J.R. (1985) Biochemistry24, 6245-6252. 7 Buck, D- and Guest,J.R. (1989) Binchem.J~ 260, "/37 747. 8 Bridget'.W.A.. Millan. W.A. and Buyer,P.D. (1968) Biochemis*.ry 7. 3608-3616. 9 Rhoads, D.B., Laimins. L. and Epstein, W. (1978) J~ Bacteriol. 135. 445-452. l0 Schauder, B.~ BlOcker, H.. Frank. IL and McCarthy, J.E.G. (!087) Gone 52. 279-283.
t|. Manlatls, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Maau,'d. Cold SpringHarbor Laboratory, Cold Spring Harbor.New York. 12 Wolodko, W.T.. BroweJe. E.R. and Bridget, W.A. (t980) J~ B~clcriol. 143, 23l-2~7. 13 Bridget, W.A., Ramale7, R.F. and Buyer, P.D. (t969) in MetV.oas ia Enzymo]ogyVol. X111 (Loweaslein. J_M.. ed.), pp. 70-75, Academic Press, New York. 14 LaemmlL U.K. (1970) Nature 227, 680-685, I5 Wolodko. W.T., Bmwnie~ E.IL. O'ConnoL M.D. and Bridger.
452-45"/.
17 Nishigagi, 1., Chert, F.T. and Hokirt. I..E. 0974) J. BioL Chem. 249. 491t -4916. 18 Hurling,J. and Vdick, S.F. (19M) J. Biol.Chem.207, 867-878. 19 Collier, G.E. and Nishimura. J.S~ (197g) J. Biol. Chum. 2.$3. 4938-4943. 20 Mann~ CI., Hurdles, S.C. and. Nishimora, J.S. (1989) J. BioL Chem. 264, 14Y1-1460. 21 Nishimura, J.S.,Mann, CJ., Yi~arra.J.,Mitchell,T. and Horowitz, P.M. (1990} Biochemistry29, 862-865.
22 Wolodko, W.T.. James. M.N.G. and Bridger, W.A. {1984)J. Biol. Chem. 259. 5316-:~320.
