Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin

Abstract

The proteinase-catalysed synthesis of [Leu]enkephalin and [Met]enkephalin was studied kinetically. Na-t-Butoxycarbonyl-amino acids and peptides or their ethyl esters served as acyl donors, and amino acid phenylhydrazides were used as acyl acceptors. Initial-velocity measurements of oa-chymotrypsin-catalysed peptide synthesis gave rise to kinetic patterns that are compatible with a ping-pong mechanism modified by a hydrolytic branch. Initial-rate and alternative-substrate inhibition patterns for papain-controlled peptide-bond formation are consistent with a sequential ordered mechanism with the acyl donor as the obligatory first substrate. On the basis of the observed kinetic features, reaction mechanisms are proposed for chymotrypsin-and papain-catalysed peptide synthesis that inversely equal those describing the pathways of proteolysis. The respective initial-velocity expressions for bireactant systems are given, along with the numerical values of the corresponding kinetic parameters.

Subtilisin-catalysed peptide synthesis and transesterification in organic solvents

“…The catalytic constant (kcat) value for transesterification was estimated to be 70.5+13.0 min -1, while the Km values for Z-Ser-OMe and EtOH were 6.6+0.7mM and 60.3_+9.9mM, respectively. A comparable kinetic scheme has also been described for chymotrypsin during catalysis of peptide bond synthesis in water (Kullman 1984) as well as in organic media (Gaertner and Puigserver 1989). In contrast, a different kinetic behaviour was described when subtilisin was used as a suspended powder to catalyse transesterification in organic solvents (Zaks and Klibanov 1988a).…”

Successful Application of the Lipase as a Catalyst in the Combinative Approach, Using an Efficient and Selective Iterative Procedure for the Chemo-Enzymatic Alpha-Amino Group Protection in the Natural Amino Acids, Representing the Ornithyl Model System as a Starting Research Pattern: Homogeneous and Heterogeneous Synthesis

“…This allows for the shifting of the reaction equilibrium towards the formation of the product. Usually, the precipitated product is collected by filtration of the reaction medium, which consists besides the product and the enzyme, also the unreacted excess from one of the components, which usually discards [15][16][17][18][19]. This methodology suffers from many disadvantages, due in many cases to the expensive reagents (costliness of the acyl or amino components), composing the related"pool" which means, that enzyme synthesis cannot be carried out in the presence of the observed low nucleophilic or electrophilic specificity, that would require the presence of a large excess from one, or from the another of the components (nucleophilic (amino) or electrophilic (acyl) compounds), which after remains unused.…”

γ-Glutamyltranspeptidase-catalysed acyl-transfer to the added acceptor does not proceed via the ping-pong mechanism

“…proteases concomitant transfer and hydrolysis occurs with the acceptor reacting with the acyl-enzyme only (Bender et al, 1964;Berezin et al, 1973;Fersht et al, 1973;Kullmann, 1984;Petkov and Stoineva, 1984;Kasche, 1984, 1985;Jakubke, 1986, 1991;Bizzozero et al, 1988;Schellenberger et al, 1990Gololobov et al, 1990Gololobov et al, , 1992Gololobov et al, , 1993. Competitive inhibition of donor consumption by the acceptor, i.e.…”