Comparison of the kinetics of the papain-catalyzed hydrolysis of glycine- and alanine-based esters and thiono esters

The kinetic constants for the papain-catalyzed hydrolysis of a series of substrates with glycine or alanine in the P1 position are discussed. The substrates have N-benzoyl, N-(p-nitrobenzoyl), N-(beta-phenylpropionyl), or N-(methyloxycarbonyl)phenylalanine attached to the P1 moiety, and kinetic constants are obtained for both esters and thiono esters. The results for the hydrolysis of esters can be readily interpreted in terms of the known specificity of papain. For any glycine ester the change in kcat/Km upon substituting C=S for C=O or upon substituting an alpha-CH3 group is minimal. However, upon making both these substitutions, i.e., going from a glycine ester to an alanine thiono ester substrate, larger changes are seen for this ratio. Data for N-benzoyl- and N-(beta-phenylpropionyl)glycine and -alanine methyl thiono esters show that k2 is the parameter most affected by the double C=S and alpha-CH3 substitution. A further conclusion is that the deacylation rate constants for any pair of glycine and alanine dithioacyl papains are similar; e.g., for the intermediates based on the "good" substrates PheAla and PheGly k3 differs by only 20%. This is a surprising finding in light of the very different conformations and interactions of the bound acyl groups revealed by resonance Raman spectroscopy and raises the possibility that specific stereochemical effects, such as the oxyanion hole and general base catalysis, are not operating in the hydrolysis of dithioacyl papains.     

The pH dependence of pre-steady-state and steady-state kinetics for the papain-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester

Pre-steady-state and steady-state kinetics of the papain (EC 3.4.22.2)-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester (ZGlyONp) have been determined between pH 3.0 and 9.5 (I = 0.1 M) at 21 +/- 0.5 degrees C. The results are consistent with the minimum three-step mechanism involving the acyl X enzyme intermediate E X P: (Formula: see text). The formation of the E X S complex may be regarded as a rapid pseudoequilibrium process; the minimum values for k+1 are 8.0 microM-1 s-1 (pH less than or equal to 3.5) and 0.40 microM-1 s-1 (pH greater than 6.0), and that for k-1 is 600 s-1 (pH independent). The pH profile of k+2/Ks (= kcat/Km; Ks = k-1/k+1) reflects the ionization of two groups with pK' values of 4.5 +/- 0.1 and 8.80 +/- 0.15 in the free enzyme. The pH dependence of k+2 and k+3 (measured only at pH values below neutrality) implicates one ionizing group in the acylation and deacylation step with pK' values of 5.80 +/- 0.15 and 3.10 +/- 0.15, respectively. As expected from the pH dependences of k+2/Ks (= kcat/Km) and k+2, the value of Ks changes with pH from 7.5 X 10(1) microM (pH less than or equal to 3.5) to 1.5 X 10(3) microM (pH greater than 6.0). Values of k-2 and k-3 are close to zero over the whole pH range explored (3.0 to 9.5). The pH dependence of kinetic parameters indicates that at acid pH values (less than or equal to 3.5), the k+2 step is rate limiting in catalysis, whereas for pH values higher than 3.5, k+3 becomes rate limiting. The observed ionizations probably reflect the acid-base equilibria of residues involved in the catalytic diad of papain, His159-Cys25. Comparison with catalytic properties of ficins and bromelains suggests that the results reported here may be of general significance for cysteine proteinase catalyzed reactions.     

The mechanism of acidic hydrolysis of esters explains the HDV ribozyme activity

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme that catalyzes the site-specific trans-esterification reaction. Using high hydrostatic pressure (HHP) technique we showed that HDV ribozyme catalyzes the reaction of RNA cleavage in the absence of magnesium ions according to mechanism of acidic hydrolysis of esters. HHP induces changes of water structure, lowering pH and effect ribozyme catalytic site structure formation without magnesium. HHP, similarly to magnesium ion at ambient pressure stabilizes the higher order RNA structure of HDV, but Mg²⁺ is not involved in the catalysis. Our results clearly support the new mechanism of HDV hydrolysis and show advantages of using HHP in analysis of macromolecules interaction.     

Catalysis by human leukocyte elastase: III. Steady-state kinetics for the hydrolysis of p-nitrophenyl esters

Steady-state kinetic parameters were determined at pH 7.4 and 25 degrees C for the human leukocyte elastase-catalyzed hydrolysis of several N-carbobenzoxy-L-amino acid p-nitrophenyl esters. The substrate specificity for these esters was quite broad, and included the Gly, Phe, and Tyr derivatives. Together with reports of a much narrower P-1 specificity for peptide-based substrates, these results suggest that interactions remote from the scissle bond between enzyme and substrate regulate primary specificity. Also, it was found that kc and kc/Km did not exhibit the same dependence on substrate structure. This is interpreted to suggest that there are significant differences in P-1 specificity between acylation and deacylation for leukocyte elastase-catalyzed reactions.     

Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.     

Antibody-enhanced hydrolysis of steroid esters

Testosterone-1α-carboxyethyl thioether was conjugated through an ester bond with the fluorescent compound umbelliferone. Testosterone-1α-carboxyethyl thioether umbelliferone conjugate was devoid of fluorescence, but yielded a fluorescent product upon incubation with hog liver esterase or with the IgG fraction of a rabbit antiserum that binds 5α-dihydrotestosterone and testosterone with similar affinity (anti-dihydrotestosterone IgG). The fluorescent compound was obtained in solution after adsorbing the ester to anti-dihydrotestosterone IgG immobilized on agarose, indicating that the appearance of fluorescence was due to hydrolysis and not to formation of a stable ester antibody complex. The antibody-enhanced hydrolysis was pH and temperature dependent and was specific with respect to the nature of the steroid and the site of steroid-umbelliferone conjugation: normal rabbit IgG and IgG directed against heterologous steroids (e.g., cortisol or progesterone) were without effect, and anti-dihydrotestosterone IgG failed to promote the hydrolysis of testosterone-7-umbelliferone or of cortisol-21-umbelliferone esters. Moreover, the hydrolysis of testosterone-1α-carboxyethyl thioether umbelliferone conjugate by anti-dihydrotestosterone IgG was inhibited by the homologous hapten but not by unrelated steroids. Hydrolysis of steroid-umbelliferone conjugates was also promoted in a nonspecific manner by nucleophilic substances such as imidazole, tyrosine or cysteine, suggesting that the antibody effect may be due to the presence of nucleophilic residues at, or near, the combining site. The results indicate that antibody can have enzyme-like properties, but the turnover is low due to slow dissociation of the reaction product from the antibody. The quasi-esterase activity of anti-steroidal antibodies can be utilized for the development of an immunoassay for these hormones.     

Papain-catalysed hydrolysis of some hippuric esters. A new mechanism for papain-catalysed hydrolyses

1. The Michaelis–Menten parameters for the papain-catalysed hydrolysis of a number of alkyl, aryl and alkyl-thiol esters of hippuric acid have been determined. 2. For all the aryl esters and most of the alkyl esters studied, the catalytic constant, k₀, is 2–3sec.⁻¹ and most probably represents deacylation of the common intermediate, hippuryl-papain. 3. Two alkyl esters and hippurylamide, however, have catalytic rate constants, k₀, less than 2–3sec.⁻¹. It is possible to interpret all the available kinetic data in terms of a three-step mechanism in which an enzyme–substrate complex is first formed, followed by acylation of the enzyme through an essential thiol group, followed by deacylation of the acyl-enzyme. 4. The logarithm of the ratio of the Michaelis–Menten parameters, which reflect the acylation rate constant, for four aryl esters of hippuric acid studied give a linear Hammett plot against the substituent constant, σ. Arguments are presented that indicate acid as well as nucleophilic catalysis in the acylation process and that the most likely proton donor is an imidazolium ion. 5. It is suggested that this imidazolium ion is part of the same histidine residue that has been tentatively implicated in the deacylation process (Lowe & Williams, 1965b). 6. A new mechanism is proposed for the papain-catalysed hydrolysis of N-acyl-α-amino acid derivatives.     

Enzymic hydrolysis of phosphonate esters. Reaction mechanism of intestinal 5'-nucleotide phosphodiesterase.

The mechanism of bovine intestinal 5'-nucleotide phosphodiesterase was investigated by determining kinetic constants of systematically varied substrates, with emphasis on esters of phosphonic acids (which have much higer Vmax values than conventional phosphodiester substrates), and by pre-steady-state kinetics using bis(4-nitrophenyl) phosphate as substrate. The results suggest a ping-pong type mechanism, with participation of a covalent enzyme intermediate.     

Transition-state stabilization at the oxyanion binding sites of serine and thiol proteinases: hydrolyses of thiono and oxygen esters

X-ray diffraction studies suggested that the tetrahedral intermediate formed during the catalysis by serine and thiol proteinases can be stabilized by hydrogen bonds from the protein to the oxyanion of the intermediate [cf. Kraut, J. (1977) Annu. Rev. Biochem. 46, 331-358; Drenth, J., Kalk, K.H., & Swen, H.M. (1976) Biochemistry 15, 3731-3738]. To obtain evidence in favor or against this hypothesis, we synthesized thiono substrates (the derivatives of N-benzoyl-glycine methyl ester and N-acetylphenylalanine ethyl ester) containing a sulfur in place of the carbonyl oxygen atom of the scissile ester bond. We anticipated that this relatively subtle structural change specifically directed to the oxyanion binding site should produce serious catalytic consequences owing to the different properties of oxygen and sulfur if transition-state stabilization in the oxyanion hole is indeed important. In fact, while in alkaline hydrolysis the chemical reactivities of oxygen esters and corresponding thiono esters proved to be similar, neither chymotrypsin nor subtilisin hydrolyzed the thiono esters at a measurable rate. This result substantiates the crucial role of the oxyanion binding site in serine proteinase catalysis. On the basis of the similar values of the binding constants found for oxygen esters and their thiono counterparts, it can be concluded that the substitution of sulfur for oxygen significantly influences transition state stabilization but not substrate binding. The thiol proteinases papain and chymopapain react with the oxygen and thiono esters of N-benzoylglycine at similar rates. Apparently, in these reactions the above stabilizing mechanism is absent or not important, which is a major mechanistic difference between the catalyses by serine and thiol proteinases.