Deacylation Rate Constants Research Articles

High-concentration salt effects in acetylcholinesterase reactions

The influence of inorganic salts on the kinetics of the reactions of acetylcholinesterase with neutral and cationic substrates at 25°C and pH 7.5 in the range of salt concentrations from physiological ionic strength up to nearly saturated solutions has been studied. In both types of substrates, the electrolytes had no influence on the acetylcholinesterase active site acylation and deacylation rate constants—the influence of the salts appeared only in the dissociation constants of the enzyme-substrate (ES) complexes. Two mechanisms were found to account for the observed effects: the primary (electrostatic) salt effect, and the salt influence on the solubility of substrates. In addition, an inhibitory effect of some 2 : 1 and 1 : 2 electrolytes (Na 2SO 4, MgCl 2, CaCl 2, SrCl 2, BaCl 2) appeared at high concentrations. The results suggest that substrates in acetylcholinesterase-catalyzed reactions are extracted into the enzyme microphase at the stage of ES-complex formation and further substrate transformation proceeds inside the enzyme.

Papain action at neutral and alkaline pH regions: A new method for the determination of individual pre-steady-state parameters from steady-state kinetics

A new method for the determination of individual pre-steady-state parameters describing papain action is reported. This procedure has been developed from the quantitative analysis of steady-state kinetics concerning the enzymatic hydrolysis of a specific chromogenic substrate, namely N-α-carbobenzoxy-L-valine p-nitrophenyl ester. The analysis of steady-state kinetics allows: (i) determination of values of K s (the fast dissociation pre-equilibrium constant for the formation of the reversible initial enzyme-substrate complex) and k +2 (the acylation rate constant) also under experimental conditions ( .e., pH > 6.0) where usual methods, based on pre-steady-state measurements, do not hold; and (ii) experimental determination of values of k +3 (the deacylation rate constant) commonly calculated from values of catalytic parameters obtained separately under steady-state and pre-steady-state conditions. This procedure allowed experimental determination of both overall steady-state parameters and individual pre-steady-state quantities for papain catalysis, not only at acid pH values but also over neutral and alkaline pH regions (i.e., between pH 2.5 and 10.5). The effect of pH on papain action is analyzed in detail, and its possible general significance for cysteine proteinase-catalyzed reactions is discussed.

Reaction of tissue-type plasminogen activator with 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride

It has recently been reported that the fluorogenic serine proteolytic active site titrant, 4-methyl-umbelliferyl-p-guanidinobenzoate (MUGB), cannot be employed in this capacity for tissue-type plasminogen activator (TPA) [Geiger, M., and Binder, B.R. (1987) Biochim. Biophys. Acta 912, 34–40]. Since this observation has such important ramifications in this area of research, we have studied the reaction of MUGB with recombinant (rec)TPA under a variety of experimental conditions and find that MUGB is indeed an effective titrant of rec-two chain TPA (recTCTPA) at 4°, a condition under which the deacylation rate constant is diminished to the point that acylation can be readily observed. The K S for the interaction of MUGB with recTCTPA is 43 μM–46 μM, the acylation rate constant, k 2, is approximately 3.6 min −1–4.2 min −1, and the rate constant for deacylation of p-guanidinobenzoyl-recTCTPA is 0.084 min −1–0.110 min −1. This same recTCTPA, after treatment with diisopropylfluorophosphate, does not react with MUGB. Single-chain TPA (recSCTPA) has been found to acylate more slowly than its two-chain counterpart and to exhibit a higher degree of turnover of the acyl-enzyme with this reagent. These results demonstrate that the active site concentration of TCTPA can be accurately determined by titration with MUGB, a consideration which is essential to the proper kinetic evaluation of this agent and its genetic variants. On the other hand, the presteady state kinetic characteristics for MUGB toward SCTPA are not favorable for its use as a titrant with this form of the enzyme.

A comparison of dogfish and bovine chymotrypsins

Bovine and dogfish chymotrypsins were compared to determine if chymotrypsin from a poikilothermic organism (spiny dogfish ( Squalus acanthias)) adapted to low temperatures possessed catalytic properties different from those of the same enzyme from a warm-blooded animal. An improved procedure was developed for isolating dogfish pancreatic chymotrypsin. The least hydrophobic and smallest substrate used, p-nitrophenyl acetate, had similar enthalpies of association ( ΔH a ) with both enzymes, whereas larger, more hydrophobic substrates had ΔH a values that were of opposite sign for the two enzymes. As the temperature increased, the association constants ( 1K s ) for p-nitrophenyl valerate and p-nitrophenyltrimethyl acetate increased for dogfish chymotrypsin and decreased for bovine chymotrypsin, while the free energies of association ( ΔG a ) remained relatively constant. Acylation of chymotrypsin was 1.5–2.5 times slower in the dogfish enzyme than in the bovine enzyme except below 15 °C with p-nitrophenyltrimethyl acetate. ΔH‡ for acylation by p-nitrophenyltrimethyl acetate were 2.0 kcal/ mol for the dogfish enzyme and 10.2 kcal/mol for the bovine, whereas ΔH‡ values were only slightly lower in the dogfish enzyme for the other two substrates. For all substrates, the deacylation rate constant ( k cat ) was greater with dogfish chymotrypsin than bovine. However, the free energies of activation ( ΔG‡) for deacylation were nearly equal between the two enzymes for each of the substrates.

ACCELERATED DEACYLATION OF ACYLATED INTERMEDIATE BY HYDROXYL FUNCTIONALIZED SURFACTANT MICELLES

Abstract The rate constants of both acylation and deacylation processes in the hydrolysis of p-nitrophenyl esters by dipeptide catalysts have been determined in the presence of hydroxyl functionalized surfactant micelles. The functional surfactants enhance the deacylation rate constants and the large rate differences are observed between the dipeptide catalysts which have the reverse sequence of amino acid residues.

Effect of monovalent cations on the pre-steady-state kinetic parameters of the plasma protease bovine activated protein C.

Activated bovine plasma protein C (APC) was not reactive with the substrate p-nitrophenyl p-guanidinobenzoate (NPGB) in the absence of cations. In the presence of increasing concentrations of Na+, the acylation rate constant, k2,app, at 7 degrees C, progressively increased from 0.32 +/- 0.03 s-1 at 12.5 mM Na+ to 1.15 +/- 0.10 s-1 at 62.5 mM Na+. A linear dependence of the reciprocal of k2,app with [Na+]-2 was observed, indicating that at least two monovalent cation sites, or classes of sites, are necessary for the catalytic event to occur. From this latter plot, the k2,max for APC catalysis of NPGB hydrolysis, at saturating [Na+] and [NPGB], was calculated to be 1.21 +/- 0.10 s-1, and the Km for Na+ was found to be 21 +/- 3 mM. The dissociation constant, Ks, for NPGB to APC, at 7 degrees C, was not altered as [Na+] was increased, yielding a range of values of 18.5 X 10(-5) to 19.9 X 10(-5) M as [Na+] was varied from 12.5 to 62.5 mM. The deacylation rate constant, k3, for p-guanidinobenzoyl-APC hydrolysis was also independent of [Na+], with a value of (3.8 +/- 1.0) X 10(-3) s-1 in the absence of Na+ or in the presence of concentrations of Na+ up to 200 mM. Identical kinetic behavior was observed when Cs+ was substituted for Na+ in the above enzymic reaction. The pre-steady-state kinetic parameters were calculated according to the same methodology as described above.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine modification in elastase. Effect on catalytic activity and conformation of the calcium-binding site.

Chemical modification of 2 +/- 0.5 arginine residues of porcine pancreatic elastase by 1,2-cyclohexanedione leads to an 85 +/- 5% loss of activity with the specific substrate N-succinyltrialanine p-nitroanilide. Modification of additional arginines does not completely abolish the enzyme activity. The modification reaction is very fast (second order rate constant = 0.24 M-1 S-1) and involves only arginine residues. Acetyltetraalanine or trifluoroacetyltetraalanine decreases the rate of cyclohexanedione-induced inactivation of the enzyme but does not significantly change the number of modified arginine residues. Other dicarbonyl reagents, butanedione or phenylglyoxal, also react with elastase but at much lower rates. Cyclohexanedione-modified elastase is partially active against a series of synthetic substrates of varying chain length. The partial inhibition results from a 2- to 5-fold increase in Km while kappa cat is increased for most substrates. For N-succinyltrialanine p-nitroanilide both the acylation and deacylation rate constants are decreased. The Ki values of a series of acetylated and trifluoroacetylated inhibitors increase 2- to 5-fold. Modified elastase is still able to react with fibrous elastin and with plasma alpha 1-proteinase inhibitor but at significantly lower rates. Modification of one arginine residue alters the properties of the calcium-binding site of elastase as demonstrated by terbium luminescence experiments. The affinity of enzyme for terbium is decreased by a factor of 10 and the circularly polarized luminescence spectrum of the terbium-elastase complex is considerably flattened. Modification of further arginine residues does not increase the extent of these alterations. Circular dichroism shows that the overall conformation of elastase is not altered following arginine modification. We speculate that the two residues modified by cyclohexanedione are Arg 65, located at about 8 A from the metal ion-binding site, and Arg 217A, located at the S'3 subsite of elastase.

Steady state kinetic evidence for an acyl-enzyme intermediate in reactions catalyzed by bovine spleen cathepsin B.

Cathepsin B from bovine spleen was shown to catalyze transacylation reactions between esters of N-substituted amino acids and nucleophiles. These reactions appeared to proceed through an intermediate between cathepsin B and the acyl portion of the substrate. Of the various nucleophiles tested, dipeptides were found to be the most effective acyl group acceptors. A method was devised for calculating the acylation and deacylation rate constants from increases in the maximum velocity of disappearance of the substrate with increasing concentrations of the nucleophile. The values for the second order rate constants for the reaction of the acyl-enzyme with the nucleophile, k4, were found to depend on the identity of the dipeptide, while the first order rate constants for formation and hydrolysis of the acyl-enzyme, k2 and k3, were dipeptide-independent. With N alpha-benzyloxycarbonyl-L-lysine p-nitrophenyl ester at pH 6.5, k2 and k3 were found to be 360 s-1 and 6.6 s-1, respectively, indicating that the deacylation step was rate-determining for the hydrolysis of this substrate. In contrast, dipeptide nucleophiles did not significantly accelerate the cathepsin B-catalyzed cleavage of either the p-nitroanilide or the 2-naphthylamide of N alpha-benzoylarginine, suggesting that the hydrolysis of these amide substrates was acylation rate-limiting. These findings support the suggestion that cathepsin B is mechanistically similar to the cysteine proteinase papain.

A novel chiral microenvironmental probe at the active site of trypsin. Extrinsic cotton effects of acyl-trypsin possessing an enantiomeric pair of chromophores.

p-Amidinophenyl esters of an enantiomeric pair of N-(2,4-dinitrophenyl)alanine (N2Ph-Ala) were both efficiently hydrolyzed by trypsin. The acylation and deacylation rate constants for the D-isomer are 1/2.5 of those for the L-isomer. Slow rates of deacylation of the two substrates made it possible to prepare the pair of enantiomeric acyl-trypsins. Circular dichroic (CD) spectra of the purified acyl-trypsins revealed that the two extrinsic chromophores are somewhat differently oriented in the chiral environment of the active site, although both chromophores could couple intermolecularly with similar intrinsic chromophores near the active site. When p-amidinophenol was added, not only was the deacylation rate of N2Ph-DAla-trypsin noticeably increased, but also the transient CD spectrum of the enzyme derivative changed markedly in comparison with that of the L-derivative. The observations indicate that the two enantiomeric acyl groups at the active site are situated in different microenvironments.

A kinetic evaluation of activated bovine blood coagulation factor IX toward synthetic substrates.

The Vmm (&7%) of bovine Factor IXas (6.9 f 0.4 pmol of P-benzoyl-L-arginine ethyl ester (BAEE) cleaved min mg of protein active sites), is increased to 7.9, 8.7,8.7,8.7, and 9.8 in the presence of 10 lll~ Ba2+, M&+, Sr2+, and Ca2+, respectively, at 30°C. The K,,, of BAEE for Factor Eas (20 f 2.5 m) is not affected by the presence of the above divalent cations. A previous study (Byrne, R., and Castellino, F. J. (1978) Arch. Biochem Biophys. 190, 687-692) has shown that the same metal ions cause a qualitatively similar change in the V,, of bovine Factor Ea, toward this substrate. Benzamidine hydrochloride was found to be a competitive inhibitor of the BAEE esterase activity for these enzymes with a KI of 3.3 f 0.3 nm toward Factor IXap This value is not altered by the presence of 10 lll~ Ca. The KI of this inhibitor for Factor IXa, was 2.9 f 0.3 nm in the presence of 10 nm Ca2+ and 6.3 f 0.5 mM in the absence of Caz+. fie-steady state kinetics, using p-nitropheny1-p'guanidinobenzoate (NphBzoGdn) as the substrate, has also been determined for Factors IXas and Ea,, also at 30°C. In the absence of Ca2+, the acylation rate constant for Factor Eas is 0.10 f 0.02 s-l, and the corresponding value for Factor IXa,is 0.11 f 0.02 s-'. These values are essentially unchanged when 10 lll~ Ca2+ is added to either assay mixture. The binding (dissociation) constant (&) of NphBzoGdn to Factor Eaa was found to be 4.4 f 1.1 X M. The KS for NphBzoGdn to Factor IXa, was determined to be 5.2 f 1.3 X M. In both cases, the Ks values were not affected by the binding of Ca2+ to the enzymes. The deacylation rate constant, ks, for this substrate with Factor Ea@ is 3.0 f 0.5 X lo-' s-' and that for Factor IXa, is 8.7 f 1.0 X lo-' s in the absence of Ca2+. These constants were found to be 6.0 k 0.7 X lo-' M and 1.0 f 0.1 X lo-' M for these same enzymes, respectively, in the presence of 10 nm Ca2+. Factor IX is a zymogen which participates in the intrinsic blood coagulation cascade and is the protein which is functionally deficient in hemophilia B. The zymogen, as isolated from bovine plasma, possesses a molecular weight of 55,400 (l), in a single polypeptide chain. Prior to participation in the intrinsic pathway, Factor IX undergoes activation, to Factor IXa, as a consequence of proteolytic cleavages in the Factor IX molecule. One activator of Factor IX is the serine protease (2) Factor XIa in the presence of Ca2+ (3,4). The Factor XIa

Multiple forms of carboxypeptidase Y from Saccharomyces cerevisiae. Kinetic demonstration of effects of carbohydrate residues on the catalytic mechanism of a glycoenzyme

In the course of our further investigation of the active site titration of carboxypeptidase Y, using 4-nitrophenyl trimethylacetate, we have found that carboxypeptidase Y can be isolated in different molecular forms. Carboxypeptidase Y obtained from Fleischmann baker's yeast has a molecular weight of 53,000, as compared to 64,000 for an enzyme species isolated from Anheuser-Busch baker's yeast. The amino acid analyses of both enzymes were essentially identical and very similar to those reported by others. However, we have found that the molecular weight difference is due to a variation in carbohydrate content as determined by gas chromatography. When carboxypeptidase Y was isolated from a single source, Anheuser-Busch baker's yeast, we observed a smaller variation in carbohydrate content. In all cases, sugar analyses revealed only mannose and N-acetylglucosamine to be present. The effect of the enzyme's carbohydrate content on the "burst kinetics" of the 4-nitrophenyl trimethylacetate reaction has been examined. In general, the Anheuser-Busch enzyme, containing more carbohydrate than the Fleischmann enzyme, reacts with a larger apparent bimolecular rate constant, kcat/Km. On the other hand, the deacylation rate constant, k3, is affected only slightly.

Effects of substituents on the rates of deacylation of substituted benzoyl papains. Role of a carboxylate residue in the catalytic mechanism.

The effect of ring substituents on the rates of deacylation of 8 meta- and para-substituted benzoyl papains was evaluated. The rate constants were found to depend upon a single ionizing group of pKa = 4.2--4.3, and to decrease by a factor of approximately 2.2 when measured in 94% D2O/H2O. The rates of deacylation are increased greatly by electron-withdrawing groups on the benzene ring. The Hammett rho value is 2.74 +/- 0.32. A plot of the rate constants for deacylation of the benzoyl papains against the corresponding constants for substituted benzoyl chymotrypsins generates a straight line of slope 1.0. This result suggests a very similar distribution of charge on the benzoyl moiety in the transition state for the two enzymes, which is interpreted in terms of the net charge of the transition state for the deacylation of nonspecific acyl papains being equal to--1 with the general base catalyzed assistance to the attack of water on the acyl enzyme being provided by the negatively charged Asp-158 rather than by the neutral Asn-175-His-159 hydrogen bond network. This result together with a survey of literature data suggests that the role of Asp-158 in papain catalysis has been underestimated. The evidence advanced to date in support of the proposition that an imidazolium-159-cysteine-25 thiolate ion pair exists in native papain is evaluated and considered to be insufficient to decide the issue.

Alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetate. Large Hammett's rho constant and participation of histidine-acylated intermediate.

A detailed examination of the mechanism of the hydrolysis of phenyl acetates by alpha-chymotrypsin [EC 3.4.21.1] was carried out. The effective deacylation rate constants of some phenyl acetates obtained by titration of the acetyl-enzyme decreased at low substrate concentrations and showed anomalous pH dependences and solvent isotope effects. The transient kinetics of deacylation of the acetyl-enzyme were biphasic. A spectrum and a breakdown rate similar to those of acetylimidazole were observed when the acetyl-enzyme was denaturated with sodium dodecyl sulfate. These results indicate the participation of histidine-acylated enzyme, which woud account for the anomalous phenomena previously found in this system, including a large value of Hammett's rho. The relation between the substrate activation and the two intermediates is discussed.

A Method for Analyzing Enzyme Kinetics with Substrate Activation and Inhibition and Its Application to the α-Chymotrypsin-catalyzed Hydrolysis of Phenyl Acetates

A general kinetic method was developed to analyze enzyme-catalyzed systems complicated by the presence of activation or inhibition by substrate. The method was applied to the alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of p-chlorophenyl and p-methoxyphenyl acetates. Deacylation rate constants which were not complicated by substrate activation were obtained. The analysis shows that the abnormal substituent dependence of kcat in the steady state hydrolysis is due not to substrate activation but to inappropriateness of the two-step mechanism or the existence of more than one acetyl-enzyme intermediate.

The Kinetics of Hydrolysis of some Extended N-Aminoacyl-L-lysine Methyl Esters by Bovine alpha and beta-Trypsins

1. The action of two active forms of bovine trypsin (alpha and beta-trypsin) on a series of specific methyl ester substrates of general formula: N-acetyl-(glycyl)n-L-lysine methyl ester (n = 0, 1, 2) and N2-benzoyl-L-arginine ethyl ester have been investigated. With the L-lysine methyl esters the catalytic rate constant for hydrolysis (kcat) was found to be significantly lower for alpha-trypsin than for beta-trypsin, whereas with N2-benzoyl-L-arginine ethyl ester there was no significant difference for the two enzymes. 2. By measurement of the kinetic constants (kcat and Km) in the presence of a nucleophile, which competes with water in the deacylation process, it has been shown that, in common with the specific ester substrates of trypsin, the rate-determining step for the extended L-lysine methyl esters is decaylation of the enzyme. 3. It has been found that by extending the aminoacyl group of N-acetyl-L-lysine methyl ester by one glycine residue (n = 1), a greatly enhanced deacylation rate constant is observed for both alpha and beta-trypsin. The higher rate constants were maintained at the higher levels by the addition of a further glycine residue (n = 2). These results have been interpreted in terms of the 'induced fit' hypothesis the substrates binding to an enzyme subsite adjacent to the active site. 4. The beta-trypsin-catalysed hydrolysis of the L-lysine substrates was investigated over a range of temperature (15--35 degrees C). The Arrhenius law was obeyed, within experimental error, by all three substrates allowing the estimation of the thermodynamic function of activation (delta S not equal to and deltaH note equal to) for the deacylation reactions. The significantly higher values of deltaS not equal to and deltaH not equal to obtained for the two extended substrates are interpreted in terms of additional hydrogen bonding between the longer aminoacyl chains and the enzyme molecule. The results are compared with those for non-extended specific substrates, which have a possible hydrophobic interaction with the enzyme surface.

Modification of isoleucine-16 acetylated delta-chymotrypsin.

Activation of acetylated chymotrypsinogen with trypsin leads to catalytically active acetylated delta-chymotrypsin containing NH2-terminal isoleucine. The importance of the cationic terminus to the control of the active conformation of acetylated delta-chymotrypsin has been demonstrated (Oppenheimer, H. L., Labouesse, B., and Hess, G. P. (1966) J. Biol. Chem. 241, 2720). Later studies appeared to suggest that the modification of isoleucine-16 of delta-chymotrypsin is not accompanied by the loss of catalytic activity as measured by the hydrolysis of N-acetyl-L-tyrosine ethyl ester (Agarwal, S. P., Martin, C. J., Blair, T. T., and Marini, M.A. (1971)Biochem. Biophys. Res. Commun. 43, 510; Blair, T. T., Marini, M. A., Agarwal, S. P., and Martin, C. J. (1971) FEBS Lett. 1486) or by the loss of active site content (Ghelis, C., Garel, J. R., and Labouesse, J. (1970) Biochemistry 9, 3902). In the present studies, controlled acetylation of the terminal alpha-aminogroup of acetylated delta-chymotrypsin with acetic anhydride led to a progressive loss of active sites of the enzyme. Determination of the catalytic and kinetic properties of the modified enzyme with the specific ester substrate N-acetyl-L-tyrosine ethyl ester or the nonspecific substrates p-nitrophenyl acetate and cinnamyol imidazole gave nearly identical results. With N-acetyl-L-tyrosine ethyl ester as substrate, the Km (app) values for acetylated delta-chymotrypsin (1.0 plus or minus 0.1 mM) and the modified enzyme (0.67 plus or minus 0.05 mM) are nearly identical and the kcat value is reduced to about 25% in the latter enzyme species. This value correlates well with about 20% of the active sites in this enzyme as measured by the rapid initial liberation of p-nitrophenol. With p-nitrophenyl acetate as substrate, the acylation rate constants (0.13 plus or minus 0.04 s(-1) at pH 6.0, 25 degrees, in 3.3% acetonitrile) and the deacylation rate constants (0.01 s(-1) at pH 8.5, 25 degrees, in 3.3% acetonitrile) are identical for the acetyl isoleucine-16 and the isoleucine-16 enzymes. Furthermore, the residual enzyme activity could be correlated well with the residual NH2-terminal isoleucine content and with the moles of [1--14C]acetyl groups incorporated per mol of the enzyme. The activity associated with the modified enzyme can be attributed to the enzyme species in which isoleucine-16 of acetylated delta-chymotrypsin is not acetylated. These data are in general agreement with the studies of Ghelis et al. (1970) but are in disagreement with the results of Blair et al. (1971) and of Agarwal et al. (1971) and confirm the hypothesis that the final conformation of acetylated delta-chymotrypsin containing an acetylated NH2 terminus is catalytically inactive and resembles acetylated zymogen in many of its physical properties.

Deacylation of Fluorine-substituted trans-Cinnamoyl-α-chymotrypsins

Deacylation of unsubstituted and of o-F-, m-F, p-F, α-F, pentafluoro-, p-methyl-, and p-trifluoromethyl-substituted trans-cinnamoyl- α-chymotrypsins has been studied from pH 4 to 8. The deacylation rate constants were found to depend upon the ionization state of a group on the enzyme with an apparent pK in the range 6.3–7.3. The hydrolysis rates of the correspondingly substituted p-nitrophenylcinnamate esters were determined at pH 10.6. Correlation of the data for these model reactions with the corresponding rates of enzyme deacylation suggests that the p-methyl-substituted acylenzyme is about an order of magnitude more reactive than expected while p-trifluoromethyl substitution results in deacylation at a rate 10 times slower than expected. The remaining substituents exert about the anticipated rate effect on deacylation.

A New Method for Determining Individual Rate Constants for Specific Non-chromophoric Substrates of Proteolytic Enzymes

Equations are developed for the pre-steady state kinetics of the proteolytic enzyme-catalyzed hydrolysis of a substrate A in the presence of a monitoring substrate (or covalent inhibitor) S of known properties. A two-intermediate acyl–enzyme mechanism is assumed in which the first intermediate is in instantaneous equilibrium with enzyme and substrate. The appearance of the first product of substrate S is characterized by two relaxation rate constants. From these constants it is possible to determine the dissociation constant and the acylation and deacylation rate constants of substrate A. Criteria are also developed for using the steady state rate parameters of A to establish conditions for which the slower relaxation process is equivalent to the deacylation rate constant of A. The technique of premixing enzyme with substrate A has certain advantages in this approach.

The Application of Insolubilized α-Chymotrypsin to Kinetic Studies on the Effect of Aprotic Dipolar Organic Solvents

Abstract Insolubilized α-chymotrypsin bound to porous glass gel has been prepared. The kinetic behavior of this fixed enzyme proved to be the same as that of the free enzyme. The effects of aprotic dipolar organic solvents on the kinetics of α-chymotrypsin-catalyzed hydrolysis were successfully studied using this insolubilized enzyme at concentrations up to 95 % v/v dioxane, taking advantage of the inability of the enzyme to aggregate. This is a considerably higher organic solvent concentration than has been achieved in homogeneous solution. The kinetic parameters of the insolubilized α-chymotrypsin-catalyzed hydrolysis of N-acetyl-l-tryptophan p-nitrophenyl ester and N-benzoyl-l-tyrosine p-nitroanilide in aqueous organic solvent medium were determined. The specific rate constant of the acylation step, κ2, does not depend on organic solvent concentration. However, the apparent Michaelis constant, Km, and deacylation rate constant, κ3, depend strongly on the organic solvent concentration. The data are discussed in terms of microscopic reversibility proposed previously for the mechanism of α-chymotrypsin action.

A kinetic study of the deacylation of alpha-benzamido-trans-cinnamoyl-chymotrypsin. Evidence for the intervention of non-enzymic species.

The apparent first-order rate constant (kobs) for the deacylation of α-benzamido-trans-cinnamoyl-chymotrypsin, determined by direct observation at 310 nm, pH 7.9 (kobs= 0.102 s−1; K. Brockelhurst and K. Williamson [1967] Chem. Commun., 666), differs from kcat determined under the same conditions with [S]0≫ [E]0 (kcat= 0.033 s−1). This result is contrary to other evidence indicating that kcat is a measure of the deacylation rate constant. The discrepancy has been shown to be due to the presence in commercial samples of α-chymotrypsin of impurities which catalyse the deacylation reaction under the conditions used for the determination of kobs ([E]0 > [S]0). The impurities, which may be removed from enzyme solutions by gel filtration on Sephadex G-25 or G-50, have been shown to be autolysis products (presumably peptides) of α-chymotrypsin. Catalysis of deacylation results from attack of the fre α-amino groups of these peptides on the acyl-enzyme, to give N-α-benzamido-trans-cinnamoyl-peptides. The consequences of these findings must be considered in interpreting any experiment in which deacylation is observed directly at high enzyme concentration, with [E]0 > [S]0. Particularly susceptible to error are experiments designed to determine the effect of pH on the deacylation rate constant, and literature results of some such experiments are critically assessed.