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E f ect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B1 L)epcri.rrnei~tof Bs'ochems'str~y, brt;'rriversityof Ofton'a,Btfuwn, Canuch K I N YAY
Received December 9, 1974 Moore, G. J . & Benoiton, N. L. (1975) Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B. Carl. J. Biochem. 53, 747-757 The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-?he by carboxypeptidase B (CPB) are increased in the presence of the modifiers @-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, 2-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration. Examination of Lineweaver-Burk plots in the presence of fixed coilce~ltrationsof Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, K,(,,,, and k,,,, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme-modifier con~plexhas a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Ba-Gly-Lys derives from an increase in the rate of breakdown of the enzyme-substrate complex to give products. Cyclohexanol differs from Bz-Gly and Bz-Gly-GIy in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme-substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier. The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior. Moore, 6 .J. & Benoiton, N. L. (1975) Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B. Cnra. J . Biochcm. 53, 747-757 Les vitesses initiales de l'hydrolyse du Bz-Gly-Lys et du Bz-Gly-Phe par la carboxypeptidase B (CPB) sont augmentees en presence des effecteurs suivants: acide @-phCnylpropionique,cyclohexanol, Bz-Gly et Bz-Gly-Gly. L'hydrolyse du tripeptide Bz-Gly-Gly-?he est aussi activee par le Bz-Gly et le Bz-Gly-Gly, mais aucun de ces effecteurs n'active l'hydrolyse du Bz-Gly-Gly-Lys, du Z-Leu-Ala-Phe ou du Bz-Gly-acide phCnyllactique par la CPB. A concentration elevee, tous ces effecteurs, sauf le cyclohexanol, manifestent des modes de liaison causant I'inhibition. L'examen des csurbes de Lineweaver-Burk, tracees en presence de concentratioils fixes de Bz-Gly, montre que diffirents m6canismes sont h la base de l'activation de l'hydrolyse des peptides neutres et basiques par la CPB, comme le refletent les valeurs des parambtres extrapslCs, K,(,,,, et k,,,. Avec Bz-Gly-Gly-Phe, il y a activation parce que le complexe enzymeeffecteur manifeste une plus grande affinitC que l'enzyme libre pour le substrat. L'activation de l'hydrolyse de BE-Gly-Lysest causee par un accroissement du taux de degradation de complexe enzyme-substrat pour donner des produits. Plusieurs propriitis distinguent le cyclohexanol du Bz-Gly et du Bz-Gly-Gly: soil mode de Biaison avec l'un ou l'autre des substrats n'est pas inhibiteur, il active seulement I'hydrolyse des dipeptides par la CPB et il exerce uil effet plus grand sur I'hydrolyse du dipeptide basique que sur celle du dipeptide neutre. De plus, le cyclohexanol active l'hydrolyse du Bz-GIy-Lys par la CPB en augmentant et 1'affinitC de liaison entre enzyme et substrat et la vitesse de l'etape catalytique. Cet effet est diflerent de celui exerck par I'effecteur Bz-Gly. Le comportement cinCtique particulier de la CPB est remarquablement semblable a celui de la carboxypeptidase A et il est un bon indice que les deux enzymes ont des structures trks simiilaires B l'intkrieur et autour de leurs sites actifs respectifs. Pour expiiquer ce comportement cinitique, on suppose que la CPB posskde un site de liaison pour recevoir les molCcules activatrices et ce site serait situ6 plus bas que l'ouverture du site actif. [Traduit par le journal] 'Supported by a grant from the Medical Research Council of Canada. 2Present addeess: Department of Pharmacology and
Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 1N4. "ssociate of the Medical Research Council of Canada.
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C.4N. J . BIOCMEM.
VOL. 53,
I975
(crystallized from methanol-ether, 86:, y~eld, m.p. 174-175 @) was hydrogenated over loC: palladium on Pancreatic CPB"(E@ 3.4.2.2) catalyzes the charcoal to give Bz-Gly-Gly-Lys. which was crystallized hydrolytic cleavage of a basic amino acid from from methanol-ether, and then water-dioxane. The com~ the @-terminus of a peptide (2). N-Acyl dipeptide pound (84'4 yield) had m.p. 211-215 "C dec, [ a ] ~-?3.6O ( c , 2 in AcOH), and contained 0.5' of Gly-Lys (detersubstrates usually have been enaployed in the mined with the amino acid analyzer). characterization of the specificity of the enzyme, Bz-Gly-Glv-Phe (crystallized from 50:, aqueous ethand CPB shows considerable activity toward anol, m.p. 217-218 "C (literature m.p. 218-219 'C (13); ' ~ (c, 1 in AcOH)) was prepared by coupling neutral peptides of this type (3-7) and ioward an [ a ] ~-+29.2c equivalent depsipeptide ($, 9). Sigmoid satura- Bz-Gly-GIy with Phe-OEt.HCI using EEDQ, followed saponification as described above. tion curves, indicative of substrate a~tivation,~byZ-Leu-Alcc-Phe: EEDQ (0.54 g, 2.2 mmol) was added to have been observed for the porcine CPB a mixture containing Phe-OEt *PIC1(0.46 mg, 2 mmol), catalyzed hydrolysis of neutral N-acyl dipeptide triethylamine (0.28 ml, 2 mmol), and Z-Leu-Ala (0.67 g, 2 mmol) in chloroform. After 12 h, the solution was substrates (6). with dilute acid, aqueous bicarbonate, dried, the This paper describes an attempt to elucidate washed solvent evaporated off, and the product was saponified the factors causing the cooperative effects ob- as described above. The product, isolated by extraction served for the cleavage of neutral N-acyl dipep- from an acidified mixture. was crystallized from ethanoltides by CPB, from a study of the effects of water. Yield 0.76 g (797;), m.p. 189-190 "C. Analysis of several modifiers on the kinetic parameters for the deprotected product indicated the presence of small amounts of three other components, presumably isomers cleavage of both neutral and basic peptides. arising from racemization occurring during coupling and These modifiers were p-phenylpropionic acid, saponification. Bz-GIj.-Pitl was prepared from L-8-phenyllactic acid cyclohexawol, Bz-Gly and Bz-Gly-Gly. For the hydrolysis of basic substrates by CPB, Bz-G%y- and 2-phenyl-5-oxazolone by a described method (14). product was crystallized as its dicyclohexylamPhe and Bz-Gly-Gly-Phe also were used as The monium salt. Yield 72' ;, m.p. 166.5-167 "C, modifiers. -2.5" (r, 1 in AcOH). For the enzyme studies the acid was regenerated by neutralization of the salt with 0.5 A' Materials H?SOdrand extracted into ethyl acetate. Cyclohexanol was purchased from Fisher Scientific, 8-phenylpropionic acid from ICN-K & K Laboratories. Cleveland, Ohio, and Gly-Gly-Phe from Schwarz-Mann. Porcine CPB (CPB-COBC. batch 9JA, 190 units/mg) This tripeptide contained no free amino acids. Bz-Gly-Lys and Bz-Gly-Phe were available from previous work (6). was a Worthington Biochemicals Corp. product. The Z-Leu-Ala, m.p. 153 "C, was a gift from Dr. J. McDer- hydrolysis of peptides was carried out in 0.1 rCa N-trismott. Bz-Gly-Gly was prepared by benzoylation of (hydroxymethyl)n~ethyl-2-amhoethanesulfonic acid 0.2 M NaCl buffer, pH 8.0, at 27 ' C , and initial rates were Gly-GBy as described ( 8 8 ) . Bz-GI),-Glg--kjqswas obtained as folows: Bz-Gly-Gly- measured as previously described (6), the product being Lys(Z)-OMe (crystallized from AcOEt ; m.p. 134-1 36 @) analyzed with an amino acid analyzer. Rates of hydrolysis of the ester Bz-Gly-Phl were was prepared (8OP: yield) by coupling Ba-GIy-Gly with Lys(Z)-OM@MCl (I 2) using N,Nr -dicyclohexylcarbodi- monitored in a pH stat (Radiometer PHM 26c/TTlla/imide. This ester bcas saponified by shaking for 30 SBUI/'SBW2/'TTA3 with a special slow delivery t u b ) in min in a mixture containii~g1.0 equiv of N NaOH and an thermostatted reaction vessels at 27 "C. Continuous equal voIume of tetrahydrofuran. The protected acid titration with a 0.5mI syringe charged with 0.08 N NaOH was performed under a nitrogen atmosphere. The T h e abbreviations for the amino acid and peptide reaction volume was 20 ml. derivatives are those recommended by the IUPAC-BUR Commission on Biochemical Nomenclature (1). The amino acid symbols represent the L-isomer except for glycine. Other abbreviations used: CPB, carboxypepFigure 1 shows the effect of 8-phenylpropionic tidase B ; CPA, casboxypeptidase A ; Z, benzyloxycar- acid on the rates of hydrolysis of several subbonyl; Bz, benzoyl; Bz-GBy-Phl, 0-(benzoylg1ycyl)-L-8phenyllactic acid: EEDQ, N-ethoxycarbonyl-2-ethoxy- strates by CPB. It is seen that, at low concentration, the modifier activates the hydrolysis of both 1,2-dihydroquinoline. T h e tern1 "positive cooperativity" previously used by the neutral and the basic dipeptide, but has no us in discussing this pherromenon (6) is abandoned to activation effect on the hydrolysis of the tripepconform with the definition of Seydo~lxet cll. (10) that tides or the ester in the same concentration positive cooperativity applies to an enzyme containing equivalent sites, i . ~ .a, subunit enzyme (Seydoux, F. J.: range. As the concentration is increased, inhibition of the hydrolysis of all peptides occurs, personal communication).
-
.
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MOORE A N D BENOITON: HYDROLYSlS BY CARBOXYPEPTLDASE B
FIG. I . ERect sf concentration s f 8-phenylpropionic acid 011 the initial rates of hydrolysis of substrates by CPB. Bz-Gly-Gly-Phe, 10.0 m M ; (A)Bz-Gly-Gly-Lys, 2.5 m M: (O) Bz-Gly-Phl, 2.5 m,W. [El = 7.47 X IO-TW for the peptides: 7.47 x 10-9 M for Bz-Gly-Phl.
(e)Bz-Gly-Lys, 2.5 m M ;(Lj)Bz-GBy-Phe, 10.0 m'W; (I)
FIG.2. Effect sf concentration of cyclohexanol on the initial rates of hydrolysis of substrates by CPB. All details as in Fig. 1.
0
40
80
Z - G Z ).m(
FIG. 3. EfTect of concentration of Hz-GBy on the initial rates of hydrolysis of substrates by CPB. Details as in Fig. 1.
higher concentrations of modifier being required to inhibit the basic substrate hydrolysis than the of Bz-Gly on rates of hydrcslysis of substrates is shown. Bz-Gly activates the hydrolysis of Bzneutral substrate hydrolysis. Figure 2 shows the efl'ect of cyclohexanol on Gly-Phe, Bz-Gly-Lys, and Ba-Gly-Gly-Phe, but the rates of hydrolysis of the same five substrates. apparently has no effect on Bz-Gly-Phl hydrokyThe degree of activation of substrate hydrolysis sis and inhibits Bz-Gly-Gly-Lys hydrolysis. is related linearly to cyclohexanol concentration Bz-Gly has a greater activating eRect on Bzup to 180 m M , and the rate of hydrolysis of the Gly-Phe compared to Bz-Gly-Lys, and the effect basic dipeptide is affected slightly more than the is linear on Ba-Gly-Phe up to 40 ~ n i WBz-Gly. At neutral dipeptide. Cyclohexanol has a very small high Bz-Gly concentration, there is a proactivating effect on Bz-Gly-Gly-Bhe hydrolysis, nounced inhibition of the rates of hydrolysis of and no effect on the hydrolysis of Bz-Gly-Gly- all substrates. Lys or Bz-Gly-Phl. Cyclohexanol does not inhibit Figure 4 shows the effect of Bz-Gly-Gly on the the hydrolysis of substrates even at concentra- rates of hydrolysis. Activation occurs for BzGly-Phe, Bz-Gly-Lys, and Bz-Gly-Gly-Bhe, but tions as high as 250 mM. In Fig. 3 the effect of increasing concentration inhibition occurs for Bz-GBy-Gly-Lys and Bz-
750
CAN. J. BIBCWEM. VOL. 53, L975
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5 00
3 -
150
400
8 00
8
-
p 300 < M g 280
bb8
50
2
z
;
tU
C9
0
4
I00
I
w
I U
-50 6 -106
-100 0
20
40
60
Ez-GI~-GI~) [mM)
80
FIG.4. Effect of concentration of BE-Gly-Gly on the initial rates sf hydrolysis of substrates by CPB. Details as in Fig. 1.
Gly-Phl at the same concentrations of modifier. The activation effect is greatest for Bz-Gly-Phe as substrate and apparently has sigmoid characteristics. As with the modifier Bz-Gly, there is a sudden drop off in rate at high concentrations of Bz-Gly-Gly for both neutral and basic dipeptides. Bz-Gly-Gly itself is hydrolyzed only slowly by CPB and the overall shape of the curve for activation of Bz-Gly-Phe by Bz-Gly-Gly is similar to the v against [ S ] curve obtained for BzGly-Gly hydrolysis by CPB ([El = '7.5 x 10-4M ) (15). . . In Fig. 5 are shown the effects of the two dipeptides Bz-Gly-Phe and Bz-Gly-Lys on the hydrolysis of each other. Bz-Gly-Phe causes activation of the hydrolysis of the latter (curve A), and the curve is characteristically sigmoid. In contrast, Bz-Gly-Lys inhibits the hydrolysis of Bz-Gly-Phe (curve B), except when the Bz-Gly-Phe :Bz-Gly-Lys ratio is 80 :1. The curves demonstrate that both dipeptides can bind in activating mode. Contrary to a previous report (16), the hydrol~sisof Bz-G1~-L~s CPB is characterized by a LiIEweaver-Burk plot (Fig. 8) which curves slightly downward, signifying substrate activationn Furthermore, above 125 nl&f, very rapid In contrast, inhibition of the hydrolysis the of B z - G l ~ - G 1 ~ - L ~ sCPB gives an ES]/v vs. KS] plot which is linear in the
[MOD IFIER] ( m ~ FIG. 5. (A) Etfect of 52-Gly-khe as modifies on the initial rate of hydrolysis of Bz-Gly-Lys (2.5 mhf) by CPB. (B) Effect of Bz-Gly-Lys as modifier on the initial rate of hydrolysis of Bz-Gly-Phe (10.0 mM) by CPB. [El = 7.5 X 10-Vf.
substrate concentration 1-1 80 mM and from which the extrapolated kinetic parameters were Kill = 19 MM?k c a t = 64 s-I at an enzyme concentration of 7.5 X M. The hydrolysis of Bz-GIy-Gly-Phe fallows apparently normal Michaelis-Menten kinetics in the substrate concentration range 5-50 ~ I Mbut , substrate inhibition occurs at higher coizcentrations. The kinetic parameters extrapolated from the linear portion 136 mM, k,,t = 12.6 of the curve where Kn1 S-I at an enzyme concentration of 7.5 X 10-8 M e 6 The constants are of course approximations because they were obtained from measurements at substrate concentrations well below the extrapolated K,,, value. Data on the activation by other compounds of the hydrolysis of Bz-Gly-Lys by CPB appear in Table I . In addition, BzMeGLy-Lys and Bz-Gly-D-homolysine were found to be activators, and 2-Leu-Ala-Phe and GIy-Gly-Phe, inhibitors. Hn contrast to the anomalous kinetic behavior observed for the hydrolysis of Bz-Gly-
-
-
--
-
6When the enzyme concentration was 7.5 X 18-W, the [ S ] / u against [S] plot displayed substrate activation in the [S] range 1-17.5 mhf (Hill coefficient, h = 1.371, a linear in the range 20-50 mM, and substrate inhibition thereafter. The observed change in kinetics at a 10-fold higher enzyme concentration is not readily explainable on the basis of models yet proposed to explain the behavior of enzymes, and possibly illustrates a gap in current knowledge on this subject.
75 1
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MOORE AND BENOITON: HYDROLYSIS BY CARBQXYPEPTIDASE B
TABLE 8 . ERect of modifiers on the initial rate of hydrolysis of Bz-Gly-Lys by CPBa Modifier
Increase in rate ((:; )
Cyclohexansl Phenylalanine Ac-G ly Bz-Gly Bz-Phe BE-Gly-G8y Bz-Gly-Phe BE-Gly-Gly-Phe Bz-Gly-Phi BOC-Gly Z-Gly-G19 Bz-Gly-Phe Bz-Gly-Lys(Z) Bz-Gly-Lys(Me2) Bz-Gly-Lys(Me8) BE-Gly-Lys(Pr) BE-GBy-Lys(Bm8)
60 20 45 90 50
62 140 120 70 2gb 44O 250" 530 3dh 30b 34b 63"
aThe syntheses of modifiers not presented in the text are described by Moose (15) and will appear in another publication. [Modifier] = 10.0 mM, [Bz-Gly-Lys] = 2.5 mM. ~[Bz-Gly-Lys]= 10.0 m M .
Phe and Bz-Gly-Gly-Phe by CPB, the hydrolysis of 2-Leu-Ala-Phe followed apparently normal Michaelis-Menten kinetics in the concentration range studied (0.25-10 mM). Kinetic parameters obtained from an [ S ] / v against [S] plot in the substrate concentration range 0.25-2.5 mM were K,, = 1.6 mM, k,,t = 81 s-I ([El 6.2 X 10-& M). The rate of hydrolysis of Z-Leu-Ala-Phe was not increased by any inodifier, while cyclohexanol, Bz-Gly and Bz-Gly-Gly had a small inhibitory effect. The v against [S] curve for the ester Bz-GlyPhl was sigmoidal in the [S] range 0.25-2.5 m M (h = 1.56). At higher concentrations, apparently normal Michaelis-Menten kinetics were observed for the fully substrate-activated enzyme from which the derived parameters were K,,,,,,ot, = 10.6 mM, k,,tc,,t, = 80 S-l ([El = 7.5 X 1WY M). Bz-Gly-Phl inhibited (177,) the hydrolysis of Bz-Gly-Bhe when both substrates were present in equal concentration (10 mM). The efTect of each modifier on the hydrolysis of a particular substrate by CPB was examined by measuring hydrolysis rates over a substrate concentration range in the presence of a fixed concentration of modifier. Figure 4 shows the effects of cyclohexanol, Bz-GLy and Bz-Gly-Gly on the Lineweaver-Burk plot for Bz-Gly-Phe. In the absence of modifier. the plot curves
-
FIG.6. Double-reciprocal plots showing the etTect of fixed concentrations of modifiers on the hydrolysis of No Bz-Gly-Phe by CPB. [EJ = 6.2 X 10-8 ~$4. (0) modifier. The three modifiers, cyclohexanol (100 mM), Bz-Gly (40 rnA4), and Bz-Gly-Gly (40 mM) had a similar effect, represented by the curve (@), the data for cyclohexanol . TABLE 2 . Kinetic constants for the hydrolysis of Bz-Gly-Gly-Phe by CPBu in the presence of modifiers Modifierb
Knl(sPp) (113 114)
Bz-Gly Bz-Gly-Gly
138 22 II
kcat
kcat!Km(app)
12.6 6.5 4.9
295 445
(s-l)
( A P 1 s-l) 91
upward, indicating substrate activation. In the presence of modifier, the positioning of the curve is changed and the amount of curvature is decreased but not abolished completely. Thus, no kinetic parameters could be derived from this system. Higher concentrations of modifier would complicate the situation further by incorporating inhibitory modes of binding, as can be seen from Figs. 3 and 4. On the other hand, apparently normal Michaelis-Menten kinetics were observed for the neutral tripeptide Bz-GIy-Gly-Phe, both in the absence and presence of modifiers (Fig. 7). Kinetic constants -_extrapolated from doublereciprocal plots show (Table 2) that in the presence of either Bz-Gly or Bz-Gly-Gly, Kr,(,,,, is
CAN. P. BPOCHEM. VOL. 53. 1975
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TABLE 3. Kinetic constants for the hydrolysis of Bz-Gly-Lys by CPBa in the presence of modifiers
Modifier concentration (mhg)
Modifier
K~(sPD)
(mW
kcat (s-l)
kcat/Km(apy)
(M-I s-l)
C yclohexanol CyclohexanoH Cyclohexanol Bz-Gly Bz-Gly Hz-Gly Bz-Gly-G1y Bz-Gly-Phe Bz-Gly-Gly-Phe
0
81
82
(m~-')
[s3 F ~ G7.. Double-reciprocal plots showing the effect of fixed concentrations of modifiers on the hydrolysis of Bz-GBy-Gly-Phe by CPB. [El = 6.2 X 10-"M. 4 0 ) No modifier: (m)Bz-Gly. 40 rnM; ( A ) Bz-Gly-Gly, 46 mM.
decreased. Since k,,t is slightly decreased, the rate of breakdown of the enzyme-modifier-substrate complex is a little slower than the rate of breakdown of the enzyme-substrate conlplex. However, the activation phenomenon is reflected totally in the decrease in value of Km(apP,in the presence of modifier, Bz-Gly-Gly having a greater effect than Bz-Gly. With the basic dipeptide Bz-Gly-Lys as substrate, apparently normal Michaelis-Menten kinetics were observed only in the presence of modifiers (Fig. 8). The double-reciprocal plot for the substrate alone curved slightly downward, indicating substrate activation. N o kinetic par-
ameters could, therefore, be obtained in the absence of modifiers. Information could, however, be obtained by determining kinetic constants in the presence of different concentrations of modifier.' These data are given in Table 3. It is seen. that in the presence of increasing concentrations of Bz-Gly, both K,(,,,,) and k,,t as well as k,,t,"Kln(app)were increased. The latter is indicative of an increase in rate of hydrolysis due to an increase in and can be interpreted as representing a situation in which the modifier causes an increase in the rate of breakdown of the enzyme-substrate complex to give products. In contrast, cyclohexanol had yet another effect. Increasing concentrations of this modifier cause a decrease in K,,(,,,,,an increase in kcat, and a resultant increase in k,,t,/K,l,!,,,,, The results reveal that activation is caused by almost equal effects on K&c,I,p,and kcat. Thus, cyclohexanol activates the hydrolysis of Bz-Gly-Lys by CPB by increasing both the binding affinity for the substrate and the rate of the catalytic step.
,.
Discussion The kinetics for the hydrolysis of N-acyl dipeptides by CPB are complicated by the ability 'Although the double-reciprocal plots for the hydsolysis of Bz-6ly-Lys in the presence of fixed concentrations of modifiers appear to be linear (Fig. 81, this cannot be the exact situation because linear; ty is possible theoretically only at saturating concentrations of modifier. However, practically speaking. any substrate activation which may occur in the presence of modifiers is undetectably small and has been disregarded for the purpose described herein, although the possibility exists that the extrapolated parameters (Table 3) may be compound terms.
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MOORE A N D BENOITON: HYDROLYSIS BY CAHBOXYPEPTIDASE B
FIG. 8. Double-reciprocal plots showing the effect of fixed concentrations of modifiers on the hydrolysis of Bz-Gly(C) No modifier; (A) Bz-G1y-Gly-Phe, 12.5 mA4; (m) Bz-Gly. 25 m:W; (a)BzGly-Phe, 25 m M ; ( A ) Bz-Gly-Gly, 25 m M ; (K)cyclohexanol, 25 mM. Lys by CPB. [El = 6.2 X 10-8 M .
of these small peptides to bind in multiple modes. The results described herein show that small pptide substrates as well as other compounds such as Bz-Gly and cyclohexanol are capable of binding in activating mode. Multiple modes of binding of substrates to CPB, giving rise to inhibition (9) and activation (a), have been reported previously. Similar effects have been observed with the enzyme CPA, which bears close structural resemblance to CPB (17). CPA is activated also in the presence of various alcohols, Bz-Gly and Bz-Gly-Gly, though this activation is limited solely to the hydrolysis of hi-acyl and hi-alkyloxycarbonyl dipeptide substrates (13). Di- and tripeptides also have been found to activate the hydrolysis of M-acyl dipeptides by CPA, and it has been shown that the activation arises because the enzyme-modifier complex has a higher affinity than the free enzyme for the substrate (18).
The results herein show that, contrary to the results reported by Zisapel and SokoIsvsky (9)
for Bz-Gly-Arg as substrate, the effect of Pphenylpropionic acid on the hydrolysis of BzGly-Eys is not straightforward (Fig. 1). At appropriate concentrations, P-phenylprogionic acid acts as an activator or an inhibitor of the hydrolysis of Bz-Gly-Lys or Bz-Gly-Phe. The relative concentrations required for the inhibitions suggest that it is of a competitive nature, since it is known that Bz-Gly-Lys binds much more strongly than Bz-Gly-Phe to the enzyme's catalytic site (6, 15). P-Phenylpropionic acid has no activation effect on the benzoylated tripeptides, or on the ester Bz-Gly-Phl, suggesting that these substrates either prevent binding of the modifier or are unaffected by the presence of the modifier bound to the enzyme. With CPA (19). p-phenylpropionic acid gives rise to mixed inhibition, competitive and non-competitive, which is related probably to the ability of the inhibitor to bind in two functional sites, as demonstrated by X-ray crystallography (20). Cyclohexanol binds only in activating mode with CPB, affecting significantly only the hy-
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754
CAW. J.
BBOCHEM.
drolyses of dipeptides. Bz-Gly and Bz-Gly-Gly, on the other hand, activrrted the hydrolysis of the neutral tripeptide as well, and moreover, at high concentrations, inhibited the hydrolysis of all the substrates, as has been shown for Bz-Gly-Lys and 2-GBy-Phe (6). The sudden inhibition at the same modifier concentration is suggestive of non-competitive inhibition, a conclusion which is supported by the observation that the hydrolysis of Bz-Gly-Gly by CPB is characterized by substrate inhibition at a substrate concentration similar to that observed to cause inhibition of the hydrolysis of the other substrates (15). The rates of hydrolysis of Bz-Gly-Gly-kys and Bz-Gly-Phl were not increased by any of the modifiers described, including peptide substrates. These, instead, bad a small inhibitory effect, or no effect at all. An analysis of the effects of modifiers on substrates from Lineweaver-Burk plots has shown that the cooperative effects present for Bz-Gly-Phe hydrolysis by CPB in the absence of modifier are reduced but not abolished in the presence of modifier (Fig. 6). However, Bz-GlyGly-$he serves as a good model compound for studies on the effects of modifiers on a neutral substrate, displaying linear Lineweaver-Burk plots in the absence and the presence of modifier (Fig. 7). For this substrate, activation occurs because the enzyme-modifier complex has a higher affinity than the free enzyme for the substrate (Table 2). Activation of the hydrolysis of substrates by CPA occurs for the same reason (1 8). The similar kinetic anomalies displayed by CPB and CPA are concordant with their close structural resemblance (17) and evidence in favor of similarities in tertiary structure at the active sites. Hn contrast to that of the neutral substrate, the hydrolysis of Bz-Gly-kys by CPB is activated by Bz-Gly for a different reason. Only the rate of breakdown sf the enzyme-substrate complex to form products appears to be affected in this system. It perhaps is not very surprising that the hydrolysis of a neutral and a basic peptide should be activated by different mechanisms by the same modifier, since the nature of the binding of these substrates at the enzyme's catalytic site must be quite digerent. Cyclshexanol has a diRerent effect than Bz-Gly on the hydrolysis of Bz-Gly-Lys by CPB. Activation by cyclshemanol arises because both the
VOL. 53, 1975
enzyme-substrate binding afinity and the rate of the catalytic step are increased. A similar effect on the kinetic parameters has been reported for the observed activation of the hydrolysis of Bz-Gly-Arg by GPB in the presence of I-butanol (21). Thus, alcohols with hydrophobic alkyl groups apparently exert a difierent effect than Bz-Gly, and probably also than the other modifier peptides employed herein, judging from the values of the kinetic constants in Table 3. It would seem that the effects exerted by Bz-Gly, Bz-Gly-Gly, Bz-Gly-Phe, and Bz-Gly-Gly-Phe are very similar, and that aliphatic alcohols present a separate case. It is significant perhaps that cycIohexano! had a greater activating effect on the basic peptide than on the neutral peptide, whereas Bz-Gly and Bz-Gly-Gly had greater efT'ects on the neutral peptide. Bt is apparent also that cyclohexanol had almost no effect on Bz-Gly-GIy-Phe hydrolysis, whereas Bz-Gly and Bz-Gly-Gly activated the hydrolysis of this substrate by CPB considerably. It is possible that alcohols bind differently than other modifiers, perhaps even at a different locus within the activator site, or even at a different activator site. However, the observed nonactivation of three different classes of substrates, Bz-Gly-Gly-Lys, 2-Leu-Ala-Phe, and BE-GlyPhl, by all modifiers including cyclohexanol, could imply that, topologically speaking, a single activator site on the CPB molecule is involved for all modifiers. The observed activation of hydrolysis of Bz-Gly-Gly-Phe only by Bz-Gly and Bz-Gly-Gly could be a special case. The hydrolysis of the more physiologically representative tripeptide Z-Leu-Ala-Bhe was not activated by modifiers. However, this may have resulted from the substrate concentration (2.5 mM) used in these studies, which was such that the enzyme may have been fully substrate-activated. Activation of Z
E f ect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B1 L)epcri.rrnei~tof Bs'ochems'str~y, brt;'rriversityof Ofton'a,Btfuwn, Canuch K I N YAY
Received December 9, 1974 Moore, G. J . & Benoiton, N. L. (1975) Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B. Carl. J. Biochem. 53, 747-757 The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-?he by carboxypeptidase B (CPB) are increased in the presence of the modifiers @-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, 2-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration. Examination of Lineweaver-Burk plots in the presence of fixed coilce~ltrationsof Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, K,(,,,, and k,,,, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme-modifier con~plexhas a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Ba-Gly-Lys derives from an increase in the rate of breakdown of the enzyme-substrate complex to give products. Cyclohexanol differs from Bz-Gly and Bz-Gly-GIy in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme-substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier. The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior. Moore, 6 .J. & Benoiton, N. L. (1975) Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B. Cnra. J . Biochcm. 53, 747-757 Les vitesses initiales de l'hydrolyse du Bz-Gly-Lys et du Bz-Gly-Phe par la carboxypeptidase B (CPB) sont augmentees en presence des effecteurs suivants: acide @-phCnylpropionique,cyclohexanol, Bz-Gly et Bz-Gly-Gly. L'hydrolyse du tripeptide Bz-Gly-Gly-?he est aussi activee par le Bz-Gly et le Bz-Gly-Gly, mais aucun de ces effecteurs n'active l'hydrolyse du Bz-Gly-Gly-Lys, du Z-Leu-Ala-Phe ou du Bz-Gly-acide phCnyllactique par la CPB. A concentration elevee, tous ces effecteurs, sauf le cyclohexanol, manifestent des modes de liaison causant I'inhibition. L'examen des csurbes de Lineweaver-Burk, tracees en presence de concentratioils fixes de Bz-Gly, montre que diffirents m6canismes sont h la base de l'activation de l'hydrolyse des peptides neutres et basiques par la CPB, comme le refletent les valeurs des parambtres extrapslCs, K,(,,,, et k,,,. Avec Bz-Gly-Gly-Phe, il y a activation parce que le complexe enzymeeffecteur manifeste une plus grande affinitC que l'enzyme libre pour le substrat. L'activation de l'hydrolyse de BE-Gly-Lysest causee par un accroissement du taux de degradation de complexe enzyme-substrat pour donner des produits. Plusieurs propriitis distinguent le cyclohexanol du Bz-Gly et du Bz-Gly-Gly: soil mode de Biaison avec l'un ou l'autre des substrats n'est pas inhibiteur, il active seulement I'hydrolyse des dipeptides par la CPB et il exerce uil effet plus grand sur I'hydrolyse du dipeptide basique que sur celle du dipeptide neutre. De plus, le cyclohexanol active l'hydrolyse du Bz-GIy-Lys par la CPB en augmentant et 1'affinitC de liaison entre enzyme et substrat et la vitesse de l'etape catalytique. Cet effet est diflerent de celui exerck par I'effecteur Bz-Gly. Le comportement cinCtique particulier de la CPB est remarquablement semblable a celui de la carboxypeptidase A et il est un bon indice que les deux enzymes ont des structures trks simiilaires B l'intkrieur et autour de leurs sites actifs respectifs. Pour expiiquer ce comportement cinitique, on suppose que la CPB posskde un site de liaison pour recevoir les molCcules activatrices et ce site serait situ6 plus bas que l'ouverture du site actif. [Traduit par le journal] 'Supported by a grant from the Medical Research Council of Canada. 2Present addeess: Department of Pharmacology and
Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 1N4. "ssociate of the Medical Research Council of Canada.
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748
C.4N. J . BIOCMEM.
VOL. 53,
I975
(crystallized from methanol-ether, 86:, y~eld, m.p. 174-175 @) was hydrogenated over loC: palladium on Pancreatic CPB"(E@ 3.4.2.2) catalyzes the charcoal to give Bz-Gly-Gly-Lys. which was crystallized hydrolytic cleavage of a basic amino acid from from methanol-ether, and then water-dioxane. The com~ the @-terminus of a peptide (2). N-Acyl dipeptide pound (84'4 yield) had m.p. 211-215 "C dec, [ a ] ~-?3.6O ( c , 2 in AcOH), and contained 0.5' of Gly-Lys (detersubstrates usually have been enaployed in the mined with the amino acid analyzer). characterization of the specificity of the enzyme, Bz-Gly-Glv-Phe (crystallized from 50:, aqueous ethand CPB shows considerable activity toward anol, m.p. 217-218 "C (literature m.p. 218-219 'C (13); ' ~ (c, 1 in AcOH)) was prepared by coupling neutral peptides of this type (3-7) and ioward an [ a ] ~-+29.2c equivalent depsipeptide ($, 9). Sigmoid satura- Bz-Gly-GIy with Phe-OEt.HCI using EEDQ, followed saponification as described above. tion curves, indicative of substrate a~tivation,~byZ-Leu-Alcc-Phe: EEDQ (0.54 g, 2.2 mmol) was added to have been observed for the porcine CPB a mixture containing Phe-OEt *PIC1(0.46 mg, 2 mmol), catalyzed hydrolysis of neutral N-acyl dipeptide triethylamine (0.28 ml, 2 mmol), and Z-Leu-Ala (0.67 g, 2 mmol) in chloroform. After 12 h, the solution was substrates (6). with dilute acid, aqueous bicarbonate, dried, the This paper describes an attempt to elucidate washed solvent evaporated off, and the product was saponified the factors causing the cooperative effects ob- as described above. The product, isolated by extraction served for the cleavage of neutral N-acyl dipep- from an acidified mixture. was crystallized from ethanoltides by CPB, from a study of the effects of water. Yield 0.76 g (797;), m.p. 189-190 "C. Analysis of several modifiers on the kinetic parameters for the deprotected product indicated the presence of small amounts of three other components, presumably isomers cleavage of both neutral and basic peptides. arising from racemization occurring during coupling and These modifiers were p-phenylpropionic acid, saponification. Bz-GIj.-Pitl was prepared from L-8-phenyllactic acid cyclohexawol, Bz-Gly and Bz-Gly-Gly. For the hydrolysis of basic substrates by CPB, Bz-G%y- and 2-phenyl-5-oxazolone by a described method (14). product was crystallized as its dicyclohexylamPhe and Bz-Gly-Gly-Phe also were used as The monium salt. Yield 72' ;, m.p. 166.5-167 "C, modifiers. -2.5" (r, 1 in AcOH). For the enzyme studies the acid was regenerated by neutralization of the salt with 0.5 A' Materials H?SOdrand extracted into ethyl acetate. Cyclohexanol was purchased from Fisher Scientific, 8-phenylpropionic acid from ICN-K & K Laboratories. Cleveland, Ohio, and Gly-Gly-Phe from Schwarz-Mann. Porcine CPB (CPB-COBC. batch 9JA, 190 units/mg) This tripeptide contained no free amino acids. Bz-Gly-Lys and Bz-Gly-Phe were available from previous work (6). was a Worthington Biochemicals Corp. product. The Z-Leu-Ala, m.p. 153 "C, was a gift from Dr. J. McDer- hydrolysis of peptides was carried out in 0.1 rCa N-trismott. Bz-Gly-Gly was prepared by benzoylation of (hydroxymethyl)n~ethyl-2-amhoethanesulfonic acid 0.2 M NaCl buffer, pH 8.0, at 27 ' C , and initial rates were Gly-GBy as described ( 8 8 ) . Bz-GI),-Glg--kjqswas obtained as folows: Bz-Gly-Gly- measured as previously described (6), the product being Lys(Z)-OMe (crystallized from AcOEt ; m.p. 134-1 36 @) analyzed with an amino acid analyzer. Rates of hydrolysis of the ester Bz-Gly-Phl were was prepared (8OP: yield) by coupling Ba-GIy-Gly with Lys(Z)-OM@MCl (I 2) using N,Nr -dicyclohexylcarbodi- monitored in a pH stat (Radiometer PHM 26c/TTlla/imide. This ester bcas saponified by shaking for 30 SBUI/'SBW2/'TTA3 with a special slow delivery t u b ) in min in a mixture containii~g1.0 equiv of N NaOH and an thermostatted reaction vessels at 27 "C. Continuous equal voIume of tetrahydrofuran. The protected acid titration with a 0.5mI syringe charged with 0.08 N NaOH was performed under a nitrogen atmosphere. The T h e abbreviations for the amino acid and peptide reaction volume was 20 ml. derivatives are those recommended by the IUPAC-BUR Commission on Biochemical Nomenclature (1). The amino acid symbols represent the L-isomer except for glycine. Other abbreviations used: CPB, carboxypepFigure 1 shows the effect of 8-phenylpropionic tidase B ; CPA, casboxypeptidase A ; Z, benzyloxycar- acid on the rates of hydrolysis of several subbonyl; Bz, benzoyl; Bz-GBy-Phl, 0-(benzoylg1ycyl)-L-8phenyllactic acid: EEDQ, N-ethoxycarbonyl-2-ethoxy- strates by CPB. It is seen that, at low concentration, the modifier activates the hydrolysis of both 1,2-dihydroquinoline. T h e tern1 "positive cooperativity" previously used by the neutral and the basic dipeptide, but has no us in discussing this pherromenon (6) is abandoned to activation effect on the hydrolysis of the tripepconform with the definition of Seydo~lxet cll. (10) that tides or the ester in the same concentration positive cooperativity applies to an enzyme containing equivalent sites, i . ~ .a, subunit enzyme (Seydoux, F. J.: range. As the concentration is increased, inhibition of the hydrolysis of all peptides occurs, personal communication).
-
.
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MOORE A N D BENOITON: HYDROLYSlS BY CARBOXYPEPTLDASE B
FIG. I . ERect sf concentration s f 8-phenylpropionic acid 011 the initial rates of hydrolysis of substrates by CPB. Bz-Gly-Gly-Phe, 10.0 m M ; (A)Bz-Gly-Gly-Lys, 2.5 m M: (O) Bz-Gly-Phl, 2.5 m,W. [El = 7.47 X IO-TW for the peptides: 7.47 x 10-9 M for Bz-Gly-Phl.
(e)Bz-Gly-Lys, 2.5 m M ;(Lj)Bz-GBy-Phe, 10.0 m'W; (I)
FIG.2. Effect sf concentration of cyclohexanol on the initial rates of hydrolysis of substrates by CPB. All details as in Fig. 1.
0
40
80
Z - G Z ).m(
FIG. 3. EfTect of concentration of Hz-GBy on the initial rates of hydrolysis of substrates by CPB. Details as in Fig. 1.
higher concentrations of modifier being required to inhibit the basic substrate hydrolysis than the of Bz-Gly on rates of hydrcslysis of substrates is shown. Bz-Gly activates the hydrolysis of Bzneutral substrate hydrolysis. Figure 2 shows the efl'ect of cyclohexanol on Gly-Phe, Bz-Gly-Lys, and Ba-Gly-Gly-Phe, but the rates of hydrolysis of the same five substrates. apparently has no effect on Bz-Gly-Phl hydrokyThe degree of activation of substrate hydrolysis sis and inhibits Bz-Gly-Gly-Lys hydrolysis. is related linearly to cyclohexanol concentration Bz-Gly has a greater activating eRect on Bzup to 180 m M , and the rate of hydrolysis of the Gly-Phe compared to Bz-Gly-Lys, and the effect basic dipeptide is affected slightly more than the is linear on Ba-Gly-Phe up to 40 ~ n i WBz-Gly. At neutral dipeptide. Cyclohexanol has a very small high Bz-Gly concentration, there is a proactivating effect on Bz-Gly-Gly-Bhe hydrolysis, nounced inhibition of the rates of hydrolysis of and no effect on the hydrolysis of Bz-Gly-Gly- all substrates. Lys or Bz-Gly-Phl. Cyclohexanol does not inhibit Figure 4 shows the effect of Bz-Gly-Gly on the the hydrolysis of substrates even at concentra- rates of hydrolysis. Activation occurs for BzGly-Phe, Bz-Gly-Lys, and Bz-Gly-Gly-Bhe, but tions as high as 250 mM. In Fig. 3 the effect of increasing concentration inhibition occurs for Bz-GBy-Gly-Lys and Bz-
750
CAN. J. BIBCWEM. VOL. 53, L975
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5 00
3 -
150
400
8 00
8
-
p 300 < M g 280
bb8
50
2
z
;
tU
C9
0
4
I00
I
w
I U
-50 6 -106
-100 0
20
40
60
Ez-GI~-GI~) [mM)
80
FIG.4. Effect of concentration of BE-Gly-Gly on the initial rates sf hydrolysis of substrates by CPB. Details as in Fig. 1.
Gly-Phl at the same concentrations of modifier. The activation effect is greatest for Bz-Gly-Phe as substrate and apparently has sigmoid characteristics. As with the modifier Bz-Gly, there is a sudden drop off in rate at high concentrations of Bz-Gly-Gly for both neutral and basic dipeptides. Bz-Gly-Gly itself is hydrolyzed only slowly by CPB and the overall shape of the curve for activation of Bz-Gly-Phe by Bz-Gly-Gly is similar to the v against [ S ] curve obtained for BzGly-Gly hydrolysis by CPB ([El = '7.5 x 10-4M ) (15). . . In Fig. 5 are shown the effects of the two dipeptides Bz-Gly-Phe and Bz-Gly-Lys on the hydrolysis of each other. Bz-Gly-Phe causes activation of the hydrolysis of the latter (curve A), and the curve is characteristically sigmoid. In contrast, Bz-Gly-Lys inhibits the hydrolysis of Bz-Gly-Phe (curve B), except when the Bz-Gly-Phe :Bz-Gly-Lys ratio is 80 :1. The curves demonstrate that both dipeptides can bind in activating mode. Contrary to a previous report (16), the hydrol~sisof Bz-G1~-L~s CPB is characterized by a LiIEweaver-Burk plot (Fig. 8) which curves slightly downward, signifying substrate activationn Furthermore, above 125 nl&f, very rapid In contrast, inhibition of the hydrolysis the of B z - G l ~ - G 1 ~ - L ~ sCPB gives an ES]/v vs. KS] plot which is linear in the
[MOD IFIER] ( m ~ FIG. 5. (A) Etfect of 52-Gly-khe as modifies on the initial rate of hydrolysis of Bz-Gly-Lys (2.5 mhf) by CPB. (B) Effect of Bz-Gly-Lys as modifier on the initial rate of hydrolysis of Bz-Gly-Phe (10.0 mM) by CPB. [El = 7.5 X 10-Vf.
substrate concentration 1-1 80 mM and from which the extrapolated kinetic parameters were Kill = 19 MM?k c a t = 64 s-I at an enzyme concentration of 7.5 X M. The hydrolysis of Bz-GIy-Gly-Phe fallows apparently normal Michaelis-Menten kinetics in the substrate concentration range 5-50 ~ I Mbut , substrate inhibition occurs at higher coizcentrations. The kinetic parameters extrapolated from the linear portion 136 mM, k,,t = 12.6 of the curve where Kn1 S-I at an enzyme concentration of 7.5 X 10-8 M e 6 The constants are of course approximations because they were obtained from measurements at substrate concentrations well below the extrapolated K,,, value. Data on the activation by other compounds of the hydrolysis of Bz-Gly-Lys by CPB appear in Table I . In addition, BzMeGLy-Lys and Bz-Gly-D-homolysine were found to be activators, and 2-Leu-Ala-Phe and GIy-Gly-Phe, inhibitors. Hn contrast to the anomalous kinetic behavior observed for the hydrolysis of Bz-Gly-
-
-
--
-
6When the enzyme concentration was 7.5 X 18-W, the [ S ] / u against [S] plot displayed substrate activation in the [S] range 1-17.5 mhf (Hill coefficient, h = 1.371, a linear in the range 20-50 mM, and substrate inhibition thereafter. The observed change in kinetics at a 10-fold higher enzyme concentration is not readily explainable on the basis of models yet proposed to explain the behavior of enzymes, and possibly illustrates a gap in current knowledge on this subject.
75 1
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MOORE AND BENOITON: HYDROLYSIS BY CARBQXYPEPTIDASE B
TABLE 8 . ERect of modifiers on the initial rate of hydrolysis of Bz-Gly-Lys by CPBa Modifier
Increase in rate ((:; )
Cyclohexansl Phenylalanine Ac-G ly Bz-Gly Bz-Phe BE-Gly-G8y Bz-Gly-Phe BE-Gly-Gly-Phe Bz-Gly-Phi BOC-Gly Z-Gly-G19 Bz-Gly-Phe Bz-Gly-Lys(Z) Bz-Gly-Lys(Me2) Bz-Gly-Lys(Me8) BE-Gly-Lys(Pr) BE-GBy-Lys(Bm8)
60 20 45 90 50
62 140 120 70 2gb 44O 250" 530 3dh 30b 34b 63"
aThe syntheses of modifiers not presented in the text are described by Moose (15) and will appear in another publication. [Modifier] = 10.0 mM, [Bz-Gly-Lys] = 2.5 mM. ~[Bz-Gly-Lys]= 10.0 m M .
Phe and Bz-Gly-Gly-Phe by CPB, the hydrolysis of 2-Leu-Ala-Phe followed apparently normal Michaelis-Menten kinetics in the concentration range studied (0.25-10 mM). Kinetic parameters obtained from an [ S ] / v against [S] plot in the substrate concentration range 0.25-2.5 mM were K,, = 1.6 mM, k,,t = 81 s-I ([El 6.2 X 10-& M). The rate of hydrolysis of Z-Leu-Ala-Phe was not increased by any inodifier, while cyclohexanol, Bz-Gly and Bz-Gly-Gly had a small inhibitory effect. The v against [S] curve for the ester Bz-GlyPhl was sigmoidal in the [S] range 0.25-2.5 m M (h = 1.56). At higher concentrations, apparently normal Michaelis-Menten kinetics were observed for the fully substrate-activated enzyme from which the derived parameters were K,,,,,,ot, = 10.6 mM, k,,tc,,t, = 80 S-l ([El = 7.5 X 1WY M). Bz-Gly-Phl inhibited (177,) the hydrolysis of Bz-Gly-Bhe when both substrates were present in equal concentration (10 mM). The efTect of each modifier on the hydrolysis of a particular substrate by CPB was examined by measuring hydrolysis rates over a substrate concentration range in the presence of a fixed concentration of modifier. Figure 4 shows the effects of cyclohexanol, Bz-GLy and Bz-Gly-Gly on the Lineweaver-Burk plot for Bz-Gly-Phe. In the absence of modifier. the plot curves
-
FIG.6. Double-reciprocal plots showing the etTect of fixed concentrations of modifiers on the hydrolysis of No Bz-Gly-Phe by CPB. [EJ = 6.2 X 10-8 ~$4. (0) modifier. The three modifiers, cyclohexanol (100 mM), Bz-Gly (40 rnA4), and Bz-Gly-Gly (40 mM) had a similar effect, represented by the curve (@), the data for cyclohexanol . TABLE 2 . Kinetic constants for the hydrolysis of Bz-Gly-Gly-Phe by CPBu in the presence of modifiers Modifierb
Knl(sPp) (113 114)
Bz-Gly Bz-Gly-Gly
138 22 II
kcat
kcat!Km(app)
12.6 6.5 4.9
295 445
(s-l)
( A P 1 s-l) 91
upward, indicating substrate activation. In the presence of modifier, the positioning of the curve is changed and the amount of curvature is decreased but not abolished completely. Thus, no kinetic parameters could be derived from this system. Higher concentrations of modifier would complicate the situation further by incorporating inhibitory modes of binding, as can be seen from Figs. 3 and 4. On the other hand, apparently normal Michaelis-Menten kinetics were observed for the neutral tripeptide Bz-GIy-Gly-Phe, both in the absence and presence of modifiers (Fig. 7). Kinetic constants -_extrapolated from doublereciprocal plots show (Table 2) that in the presence of either Bz-Gly or Bz-Gly-Gly, Kr,(,,,, is
CAN. P. BPOCHEM. VOL. 53. 1975
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TABLE 3. Kinetic constants for the hydrolysis of Bz-Gly-Lys by CPBa in the presence of modifiers
Modifier concentration (mhg)
Modifier
K~(sPD)
(mW
kcat (s-l)
kcat/Km(apy)
(M-I s-l)
C yclohexanol CyclohexanoH Cyclohexanol Bz-Gly Bz-Gly Hz-Gly Bz-Gly-G1y Bz-Gly-Phe Bz-Gly-Gly-Phe
0
81
82
(m~-')
[s3 F ~ G7.. Double-reciprocal plots showing the effect of fixed concentrations of modifiers on the hydrolysis of Bz-GBy-Gly-Phe by CPB. [El = 6.2 X 10-"M. 4 0 ) No modifier: (m)Bz-Gly. 40 rnM; ( A ) Bz-Gly-Gly, 46 mM.
decreased. Since k,,t is slightly decreased, the rate of breakdown of the enzyme-modifier-substrate complex is a little slower than the rate of breakdown of the enzyme-substrate conlplex. However, the activation phenomenon is reflected totally in the decrease in value of Km(apP,in the presence of modifier, Bz-Gly-Gly having a greater effect than Bz-Gly. With the basic dipeptide Bz-Gly-Lys as substrate, apparently normal Michaelis-Menten kinetics were observed only in the presence of modifiers (Fig. 8). The double-reciprocal plot for the substrate alone curved slightly downward, indicating substrate activation. N o kinetic par-
ameters could, therefore, be obtained in the absence of modifiers. Information could, however, be obtained by determining kinetic constants in the presence of different concentrations of modifier.' These data are given in Table 3. It is seen. that in the presence of increasing concentrations of Bz-Gly, both K,(,,,,) and k,,t as well as k,,t,"Kln(app)were increased. The latter is indicative of an increase in rate of hydrolysis due to an increase in and can be interpreted as representing a situation in which the modifier causes an increase in the rate of breakdown of the enzyme-substrate complex to give products. In contrast, cyclohexanol had yet another effect. Increasing concentrations of this modifier cause a decrease in K,,(,,,,,an increase in kcat, and a resultant increase in k,,t,/K,l,!,,,,, The results reveal that activation is caused by almost equal effects on K&c,I,p,and kcat. Thus, cyclohexanol activates the hydrolysis of Bz-Gly-Lys by CPB by increasing both the binding affinity for the substrate and the rate of the catalytic step.
,.
Discussion The kinetics for the hydrolysis of N-acyl dipeptides by CPB are complicated by the ability 'Although the double-reciprocal plots for the hydsolysis of Bz-6ly-Lys in the presence of fixed concentrations of modifiers appear to be linear (Fig. 81, this cannot be the exact situation because linear; ty is possible theoretically only at saturating concentrations of modifier. However, practically speaking. any substrate activation which may occur in the presence of modifiers is undetectably small and has been disregarded for the purpose described herein, although the possibility exists that the extrapolated parameters (Table 3) may be compound terms.
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MOORE A N D BENOITON: HYDROLYSIS BY CAHBOXYPEPTIDASE B
FIG. 8. Double-reciprocal plots showing the effect of fixed concentrations of modifiers on the hydrolysis of Bz-Gly(C) No modifier; (A) Bz-G1y-Gly-Phe, 12.5 mA4; (m) Bz-Gly. 25 m:W; (a)BzGly-Phe, 25 m M ; ( A ) Bz-Gly-Gly, 25 m M ; (K)cyclohexanol, 25 mM. Lys by CPB. [El = 6.2 X 10-8 M .
of these small peptides to bind in multiple modes. The results described herein show that small pptide substrates as well as other compounds such as Bz-Gly and cyclohexanol are capable of binding in activating mode. Multiple modes of binding of substrates to CPB, giving rise to inhibition (9) and activation (a), have been reported previously. Similar effects have been observed with the enzyme CPA, which bears close structural resemblance to CPB (17). CPA is activated also in the presence of various alcohols, Bz-Gly and Bz-Gly-Gly, though this activation is limited solely to the hydrolysis of hi-acyl and hi-alkyloxycarbonyl dipeptide substrates (13). Di- and tripeptides also have been found to activate the hydrolysis of M-acyl dipeptides by CPA, and it has been shown that the activation arises because the enzyme-modifier complex has a higher affinity than the free enzyme for the substrate (18).
The results herein show that, contrary to the results reported by Zisapel and SokoIsvsky (9)
for Bz-Gly-Arg as substrate, the effect of Pphenylpropionic acid on the hydrolysis of BzGly-Eys is not straightforward (Fig. 1). At appropriate concentrations, P-phenylprogionic acid acts as an activator or an inhibitor of the hydrolysis of Bz-Gly-Lys or Bz-Gly-Phe. The relative concentrations required for the inhibitions suggest that it is of a competitive nature, since it is known that Bz-Gly-Lys binds much more strongly than Bz-Gly-Phe to the enzyme's catalytic site (6, 15). P-Phenylpropionic acid has no activation effect on the benzoylated tripeptides, or on the ester Bz-Gly-Phl, suggesting that these substrates either prevent binding of the modifier or are unaffected by the presence of the modifier bound to the enzyme. With CPA (19). p-phenylpropionic acid gives rise to mixed inhibition, competitive and non-competitive, which is related probably to the ability of the inhibitor to bind in two functional sites, as demonstrated by X-ray crystallography (20). Cyclohexanol binds only in activating mode with CPB, affecting significantly only the hy-
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drolyses of dipeptides. Bz-Gly and Bz-Gly-Gly, on the other hand, activrrted the hydrolysis of the neutral tripeptide as well, and moreover, at high concentrations, inhibited the hydrolysis of all the substrates, as has been shown for Bz-Gly-Lys and 2-GBy-Phe (6). The sudden inhibition at the same modifier concentration is suggestive of non-competitive inhibition, a conclusion which is supported by the observation that the hydrolysis of Bz-Gly-Gly by CPB is characterized by substrate inhibition at a substrate concentration similar to that observed to cause inhibition of the hydrolysis of the other substrates (15). The rates of hydrolysis of Bz-Gly-Gly-kys and Bz-Gly-Phl were not increased by any of the modifiers described, including peptide substrates. These, instead, bad a small inhibitory effect, or no effect at all. An analysis of the effects of modifiers on substrates from Lineweaver-Burk plots has shown that the cooperative effects present for Bz-Gly-Phe hydrolysis by CPB in the absence of modifier are reduced but not abolished in the presence of modifier (Fig. 6). However, Bz-GlyGly-$he serves as a good model compound for studies on the effects of modifiers on a neutral substrate, displaying linear Lineweaver-Burk plots in the absence and the presence of modifier (Fig. 7). For this substrate, activation occurs because the enzyme-modifier complex has a higher affinity than the free enzyme for the substrate (Table 2). Activation of the hydrolysis of substrates by CPA occurs for the same reason (1 8). The similar kinetic anomalies displayed by CPB and CPA are concordant with their close structural resemblance (17) and evidence in favor of similarities in tertiary structure at the active sites. Hn contrast to that of the neutral substrate, the hydrolysis of Bz-Gly-kys by CPB is activated by Bz-Gly for a different reason. Only the rate of breakdown sf the enzyme-substrate complex to form products appears to be affected in this system. It perhaps is not very surprising that the hydrolysis of a neutral and a basic peptide should be activated by different mechanisms by the same modifier, since the nature of the binding of these substrates at the enzyme's catalytic site must be quite digerent. Cyclshexanol has a diRerent effect than Bz-Gly on the hydrolysis of Bz-Gly-Lys by CPB. Activation by cyclshemanol arises because both the
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enzyme-substrate binding afinity and the rate of the catalytic step are increased. A similar effect on the kinetic parameters has been reported for the observed activation of the hydrolysis of Bz-Gly-Arg by GPB in the presence of I-butanol (21). Thus, alcohols with hydrophobic alkyl groups apparently exert a difierent effect than Bz-Gly, and probably also than the other modifier peptides employed herein, judging from the values of the kinetic constants in Table 3. It would seem that the effects exerted by Bz-Gly, Bz-Gly-Gly, Bz-Gly-Phe, and Bz-Gly-Gly-Phe are very similar, and that aliphatic alcohols present a separate case. It is significant perhaps that cycIohexano! had a greater activating effect on the basic peptide than on the neutral peptide, whereas Bz-Gly and Bz-Gly-Gly had greater efT'ects on the neutral peptide. Bt is apparent also that cyclohexanol had almost no effect on Bz-Gly-GIy-Phe hydrolysis, whereas Bz-Gly and Bz-Gly-Gly activated the hydrolysis of this substrate by CPB considerably. It is possible that alcohols bind differently than other modifiers, perhaps even at a different locus within the activator site, or even at a different activator site. However, the observed nonactivation of three different classes of substrates, Bz-Gly-Gly-Lys, 2-Leu-Ala-Phe, and BE-GlyPhl, by all modifiers including cyclohexanol, could imply that, topologically speaking, a single activator site on the CPB molecule is involved for all modifiers. The observed activation of hydrolysis of Bz-Gly-Gly-Phe only by Bz-Gly and Bz-Gly-Gly could be a special case. The hydrolysis of the more physiologically representative tripeptide Z-Leu-Ala-Bhe was not activated by modifiers. However, this may have resulted from the substrate concentration (2.5 mM) used in these studies, which was such that the enzyme may have been fully substrate-activated. Activation of Z
