Kinetic solvent isotope effects on the deacylation of specific acyl-papains. Proton inventory studies on the papain-catalysed hydrolyses of specific ester substrates: analysis of possible transition state structures

Abstract

1. The hydrolyses of the p-nitrophenyl esters of N-benzyloxycarbonylglycine, alpha-N-benzyloxycarbonyl-L-lysine and N-methoxycarbonyl-L-phenylalanylglycine catalysed by papain (EC 3.4.22.2) have been studied in solvents having a variable composition of 2H2O and H2O. 2. kcat., which represents deacylation in the papain-catalysed hydrolysis of reactive esters, is some 2.3-fold less in 2H2O compared with H2O. The magnitude of kcat. has been determined as a function of the 2H atom fraction of the solvent. 3. Both linear and non-linear methods of least-square regression analysis have been applied to the data in order to obtain best-fit parameter values for several three-parameter models which express kcat. in terms of the 2H atom fraction of the solvent. These models represent some possible modes of restructuring of the active site protonic configuration consequent upon transition state formation. 4. The results of curve fitting reveal an essentially linear dependence of kcat. upon the 2H atom fraction, and it may therefore be concluded that the isotope effect originates from a single proton which is in the process of transfer in the transition state. 5. It is postulated on the basis of this and other evidence that the mobile proton is transferred from an attacking water molecule to the imidazole side chain of His-159 during tetrahedral intermediate formation. This has the effect of stabilizing the transition state and promoting catalysis. The role of His-159 in deacylation is therefore to provide general base catalysis. 6. Models that involve two or more protons, such as a two-proton relay system analogous to that proposed for the serine proteinases, or a multiproton 'medium' effect, are considered unlikely on the basis of the data reported in this paper. 7. A more detailed examination of possible transition state structures reveals that the only structure compatible with available experimental data and consistent with certain theoretical predictions is one in which the proton translocated in concern with reorganization of the heavy atom framework. In addition, the transition state vibrations of the mobile proton are strongly coupled to those of the heavy atoms. These properties of the transition state are also manifest in the transition state for the deacylation of serine proteinases.

Role of Glutamate-268 in the Catalytic Mechanism of Nonphosphorylating Glyceraldehyde-3-phosphate Dehydrogenase from Streptococcus mutans

“…Moreover, the rate-determining step within deacylation has been associated with the hydrolysis from the solvent isotopic effect. If the observed deacylation rate does not include any contribution from preceding steps of the acylation, and as already suggested for Cys proteases (20), assuming that the mechanistic path of deacylation includes a tetrahedral intermediate whose breakdown is not rate-limiting, then the pK a of 6.1 would not originate from Cys-302 free thiolate but would rather reflect the ionization of the hydroxyl group of this intermediate.…”

Kinetics of enzyme acylation and deacylation in the penicillin acylase‐catalyzed synthesis of β‐lactam antibiotics

“…The relation between the value of a rate constant and the mole fraction D 2 O can be described by the simplified form of the Gross-Butler equation [20],…”

Proton Transfer during Catalysis by Hydrolases

“…To investigate the possible operation of this structural feature in catalysis, solvent isotope effects and proton inventories were determined for hydrolysis of the acyl-enzymes that form during reaction of papain with the methyl esters of N-benzoyl-Gly and N-(b-phenylpropionyl)Gly, and the p-nitrophenyl esters of Z-Gly, Z-Lys, and Nmethoxycarbonyl-Phe-Gly [45,46]. To investigate the possible operation of this structural feature in catalysis, solvent isotope effects and proton inventories were determined for hydrolysis of the acyl-enzymes that form during reaction of papain with the methyl esters of N-benzoyl-Gly and N-(b-phenylpropionyl)Gly, and the p-nitrophenyl esters of Z-Gly, Z-Lys, and Nmethoxycarbonyl-Phe-Gly [45,46].…”