MECHANISTIC STUDY ON CARBOXYPEPTIDASE Y-CATALYZED TRANSACYLATION REACTIONS - MUTATIONALLY ALTERED ENZYMES FOR PEPTIDE-SYNTHESIS

In (serine) carboxypeptidase Y the recognition of the C-terminal carbo xylate group of peptide substrates is due to the side chains of Asn51 and Glu145 functioning as hydrogen bond donors. Carboxypeptidase Y mut ants, where these amino acid residues have been substituted for other residues, have been investigated for their applicability in transacyla tion reactions. It is shown that Glu145 is not important for the bindi ng of amino acid nucleophiles, consistent with the fact that at basic pH, where synthesis reactions are carried out, Glu145 cannot act as a hydrogen bond donor when deprotonated. In fact, its substitution for A la is beneficial for the yield of synthesis, an effect which is probab ly due to complete or partial elimination of the charge repulsion betw een the alpha-carboxylate group of the amino acid nucleophiles and Glu 145 creating more favorable binding modes for the nucleophiles. Replac ement of Asn51 with Ala eliminates the capacity of the enzyme to accep t amino acids as nucleophiles, suggesting that a hydrogen bond donor a t position 51 is required. Incorporation of other hydrogen bond donors at this position, i.e. Ser and Gln, shows that the distance of the bo und nucleophile to the acylated Ser146 is important for synthesis yiel ds. With Ser (long distance), Asn (wild-type), and Gln (short distance ) the yields using H-Val-OH as nucleophile were 5%, 32%, and 97%, resp ectively. On the other hand, a mutant enzyme with a Gln at position 51 results in a lower k(cat) for the hydrolysis of peptide substrates as compared to an Asn (wild-type) or a Ser at this position. Thus, short distance is favorable for synthesis and unfavorable for hydrolysis. A novel mechanism for carboxy peptidase Y-catalyzed transacylation reac tions, describing the fraction of aminolysis (fa) by the parameters fa (max) and K(N,app), is suggested. This mechanism contains the new feat ure that hydrolysis of the acyl-enzyme is possible with the leaving gr oup/nucleophile bound within the S1' binding site. It is further shown that the low yields often encountered in transpeptidation reactions a re due to attack by water on the acyl-enzyme intermediate, while the l eaving group remains bound to the enzyme. This reaction maybe suppress ed by mutational alterations of the enzyme, hence increasing its synth etic capability, in particular in amidation reactions.