Kinetic characterization of carboxypeptidase-Y-catalyzed peptide semisynthesis Prediction of yields.

Carboxypeptidase-Y-catalyzed peptide semisynthesis has been characterized at pH 7.5, 25°C from initial rate steady state kinetic and progress reaction studies of hydrolysis and aminolysis ofα-N-benzoyl-L-tyrosine 4-nitro-anilide using the natural L-amino acids and their amides as nucleophiles. The reaction mechanism previously shown to account for carboxypeptidase-Y-catalyzed aminolysis reactions (Christensen et al., 1992) was found also to account for all of the reactions studied here. It involves in addition to the classical serine proteinase mechanism: i) complex formation between the free enzyme and the nucleophile, an interaction characterized by the competitive inhibition constant,K i, and ii) reaction of the nucleophile with the acylated enzyme forming a complex of enzyme and aminolysis product, characterized by the aminolysis kinetic parameter,K' N.A competitive inhibitory effect showing binding to the free enzyme is seen mainly with large hydrophobic amino acids and their amides i.e. the same residues as those preferred on either side of the scissile bond in carboxypeptidase-Y substrates. The stoichiometry of the inhibition is 1: 1and the actual binding position most likely is that of the leaving group of substrates,S' 1.Aminolysis effects are obtained with a wide range of amino acids and amino acid amides, exceptions are Pro and, probably due to their low solubility, Tyr, Trp, Asp and Glu. TheK' N-values show relatively little dependence on the chemical nature of the side groups, but a marked difference between the amino acid and its amide. The amides interact more strongly. The kinetic parameter,k c/Km, of the hydrolysis of the aminolysis products is another important factor in peptide semisynthesis. Thek c/Km-values obtained of the amidated aminolysis products are much less than those of the products formed with free amino acids. All in all this leads to rather efficient aminolysis with the L-amino acid amides and poor aminolysis with the L-amino acids.