June 20, 1955
INHIBITORDISSOCIATION CONSTANTS OF CPCHYMOTRYPSIN
pies of activation for the resin-catalyzed hydrolysis are 13 t o 18 cal. deg.-l mole-' higher than for acid hydrolysis." Thus the greater efficiency of the resin is explained by the increase in the entropy of activation as compared with the entropy of activation of the acid-catalyzed reaction. This increase
[COSTRIBUTIUS S O . 1943
F R O M THE
3365
in the entropy of activation is probably due to the fixation of the dipeptide molecule to the resin particle. Acknowledgment.-This work was supported by a grant from the Herman Frasch Foundation. COLUMBUS,
OHIO
GATESAND CRELLIS LABORATORIES O F CHEMISTRY, CALIFORNIA
ISSTITUTE O F
TECHNOLOGY]
The Evaluation of the Enzyme-Inhibitor Dissociation Constants of &-Chymotrypsin and Several Pairs of Charged and Uncharged Competitive Inhibitors at pH 7.9 and 6 . 9 1 p 2 BY ROBERTJ. FOSTER AND CARL
NIEMANN3
RECEIVED DECEMBER 29, 1954 The enzyme-inhibitor dissociation constants of a-chymotrypsin and several pairs of charged and tincharged competitive inhibitors of this enzyme which were evaluated previously in aqueous solutions a t 25" and PH 7.9 and 0.02 11.1 in the T H A M component of a THAM-HC1 buffer now have been evaluated in aqueous solutions a t 25' and pH 6.9 and 0.2 M i n the THAM component of a THAM-HCI buffer. A comparison of these two sets of dissociation constants has led t o the suggestion t h a t the development of a negative charge in the environment of the catalytically active site of the enzyme particularly at pH 7.9 is responsible for the lesser affinity of a-chymotrypsin a t PH 7.9 than a t pH G.9 for certain negatively charged rompetitive inhibitors of this enzyme. T h e effect of adding phosphate ion to the above systems ha? been discussed.
In 19.77, Bergmann and Fruton4 reported that achymotrypsin, acting in aqueous solutions h!/15 in a phosphate buffer and a t 40" and PH 7.6-7.8, hydrolyzed carbobenzoxy-L-tyrosylglycinamide, to give carbobenzoxy-L-tyrosine and glycinamide, but did not hydrolyze carbobenzoxy-L-tyrosylglycine. On the basis of the above evidence these authors concluded that the enzyme was incapable of causing the hydrolysis of specific substrates in which a carboxyl group was bonded to the non-carbonyl carbon atom immediately adjacent to the nitrogen atom involved in the susceptible peptide bond. This conclusion was substantiated further by the subsequent report5 that a-chymotrypsin, acting in aqueous solutions M / 1 5 in a phosphate buffer,6 but a t 25" and pH 7. 1-7.5, hydrolyzed carbobenzoxyL-phenylalanylglycinamide but did not hydrolyze carbobenzoxy-L-phenylalanylglycine,and by the claim' that carbobenzoxy-L-glutamyl-L-tyrosylglycinamide and L-glutamyl-L-tyrosylglycinamide were hydrolyzed in the presence of a-chymotrypsin but that carbobenzoxy-L-glutamyl-L-tyrosylglycine was not.s In 1950, Neurath and SchwertJgrecognizing that the carboxyl group in question would be completely ionized in aqueous solutions in the region of pH 7-8, concluded, on the basis of the above evidence, t h a t the presence of a negative charge near the susceptible bond caused a loss in substrate activity. (1) Supported in p a r t b y a grant from Eli Lilly and Co.
(2) Cf. R. J. Foster, P h . D . Thesis, Calif. Inst. Tech., Pasadena, Calif., 1952. (3) To whom inquiries regarding this article should he sent. (4) M. Bergmann and J. S. Fruton, J . Bid. Chem., 118,405 (1937). (8) J. S . Fruton and M . Bergmann, ibid., 146, 253 (1942). ( 6 ) Private communication from J . S. Fruton. (7) hl. Bergmann and J . S. F r u t o n , Advances i n Enzymol., 1, 63 (1941). (8) T h e authors have been informed by J. S. Fruton t h a t these experiments were conducted in aqueous solutions a t 38-39O and p H 7 t i and .MI15 in a phosphate buffer and t h a t in the experiment with c n r b o h e n z o r y - L - g l u t a m y l - ~ - i ~ r o s y l g l ~ ~ i n ~the m i dspecific e buhstrate U a s initially present in suspension. (9) 11. Yrrirath and G. a '. S r h w r r t , Chpin. Re113, 46, 179 (1950).
The first suggestion t h a t a negative charge was present a t or near the catalvtically active site of the enzyme and that an electrostatic repulsion could arise from the interaction of this negative charge with that present in a competitive inhibitor containing a carboxylate group was offered by Neurath and Schwertg on the basis of experiments described by Kaufman and Neurathlo.'l which were conducted a t 25' and pH 7.8 in the presence of a 0.1 M phosphate buffer when aqueous solutions were employed, or a 0.045 i M phosphate buffer when the solvent system was 30y0aqueous methanol. However, the experiments of Kaufman and NeurathlO," do not provide a direct and unambiguous demonstration t h a t the affinity of a-chymotrypsin, when evaluated in aqueous solutions a t p H 7.8 and in the presence of a phosphate buffer, for a competitive inhibitor containing a negatively charged carboxylate group is substantially less than for an uncharged competitive inhibitor which possesses the same structural features except for the replacement of the negatively charged carboxylate group by an uncharged group of approximately the same volume. The only uncharged competitive inhibitors which were studied by Kaufman and Neurathlo?l1 were DL-1-phenyl-2-acetamidobutanone-3 and DL- 1p-hvdroxyphenyl-2-acetamidobutanone-3and the inhibition constants determined were for the DLmixtures. It was a tenuous comparison of these composite inhibition constants with those of aceturic acid, hippuric acid, acetyl-DL-methionine, benzoyl-DL-methionine, benzoyl-D-, L- and DL-phenylalanine, and presumably that of 0,N-diacetyl-Ltyrosine, that led Neurath and SchwertQ to the conclusion that replacement of a carboxylate group by an acetyl group results in an increased affinity of the enzyme for the inhibitor, under the conditions ~)reviouslvspecified, rather than a direct cornparison of, for example, acetyl-L-phenylalanine with (10) S K a u f m r n and H I\-eurath, A Y C Btochein, ~ 31, 2 4 7 '1949) i l l ) S Kmifman and H Weirrath, J Rzol Cheiit , 181, II?(1919)
L- 1-phenyl-2-acetamidobutanone-3 where the only TABLE I structural factor involved is the replacement of a ENZYME-~XHIEITOR DISSOCIATIOX CONSTANTS carboxylate group by an acetyl group. H a n d Na Sand G b h -A -3 - 3 In a subsequent study in which the enzyme-inInhibitor K1C.d Foj /ilc [;o/ I.'" hibitor dissociation constants of a-chymotrypsin and p- I'henvlpropionat e' 5.5 3 1 O,!l ?j&G 2 2 a relatively large number of carboxylic acids were ,%Phenylpropionamide i-L2 2.0 .. 00 * 10 1 . 7 14 2 ,: 0.8 evaluated in aqueous solutions at 2.5' and p H 7.8 -j - P hen y l hu tyra t ee i n the presence of a 0.1 AI phosphate buffer, Neu- ;-I'henvll~iityramide rath and Gladner, 12inan argument which was based upon the use of absolute values of the above dissociation constants, the binding energies reported for Revised valuesIq of Huang and Siemann'e for aqueoub the interaction of certain dye anions and homolo- solutions a t 2:' a n d PH 7.9 and 0.02 .I1in the THAM cotn\-:*lues of Neurath a n d gous alkyl sulfates with serum albumin, 1 3 b 1 * and the ponent of :L THAL>I-HCl buffer. for aqueous solutions at 25' :tnd pH 7 . 8 and 0.1 *lf behavior of an entirely different enzyme system,15 Cladner12 The value.; I n units of 10F3 -21. in a phosphate huffer. again expressed the belief that an electrostatic re- given in this column d o not differ significantly from the prrpulsion arising from the interaction of a competitive viously reported valuesI6 escept for a more realistic probablc inhibitor bearing a negatively charged carboxylate error. p Since this inhihitor was ;!dded to the reaction system in the form of it.; sodium salt the corresponding K T group with a negative charge at the catalytically value is suhject to correction for an ionic strength effect"' active site was a likely possibility even though it which in this instmce is probably Icss than the estimated was reported b y these authorsI2 that the K I values experimental error. f Values of - A F o in kcal. per mole at of P-phenylpropionate and P-phenoxyethanol were 2.7' to the ne:irest 0.1kcal. almost identical. corresponding to a change in - lF"a t 23O of approxIn 1952, Huang and Niemann16 called attention imately 1 kcal. by simplv transferring the system to the fact t h a t when the enzyme-inhibitor disso- from a 0.02 uni-univalent TH-%lI-HCl buffer to ciation constants of a-chymotrypsin and a series of a 0. I uni-pdyvnler1t phosphate buffer. That this competitive inhibitors of the type R ( C H Z ) ~ C O ? - effect is due to an electrostatic repulsion o f the were evaluated a t 2.5' and p H 7.9 in aqueous solu- negatively charged carboxylate group, present in tions 0.02 M with respect to the THAM" conipo- the bifunctional competitive inhibitors, by a neganent of a THAAl-HCl buffer, one obtained KI tive charge at or near the catalytically active sitc \ d u e s which were approximately five times greater of the eriz)rrne which is operative, in aqueous soluthan those reported by Neurath and Gladner" for tions at 25' and pH '7.0 ==I 0.1 in a 0.02 Jfuni-univnthe same competitive inhibitors but based upon an lent T H h l l - H C l buffer but which is not operative evaluation at 2.5' and PH 7.8 in aqueous solutions in a 0.1 J f uni-polyvalent phosphate buffer is sug0.1 Jl with respect to a phosphate buffer. Recently gested by the fact that in the former system the h-i we have re-evaluated the primary experimental values of the bifunctioxal competitive inhibitors data obtained by Huang and Niemann16 with the bearing a negatively charged carboxylate group are aid of the procedure described by Jennings and approxiniatel>- five, actually :3.(i-6,.5, times gre:tter Niemann18 and the revised K I values,19 ;.e., the thaii the I
