The role of Tyr248 probed by mutant bovine carboxypeptidase A: insight into the catalytic mechanism of carboxypeptidase A.

摘要: We have investigated the function of Tyr248 using bovine wild-type CPA and its Y248F Y248A mutants to find that K(M) values were increased by 4.5-11-fold k(cat) reduced 4.5-10.7-fold replacement with Phe for hydrolysis hippuryl-L-Phe (HPA) N-[3-(2-furyl)acryloyl]-Phe-Phe (FAPP), respectively. In case O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate (ClCPL), an ester substrate, value was 2.5-fold, 20-fold. The Ala decreased about 18- 237-fold HPA ClCPL, respectively, demonstrating aromatic ring plays a critical role in enzymic reaction. increases only 6- 5-fold Thus, present study indicates clearly important not binding substrate but also hydrolysis. kinetic results may be rationalized proposition phenolic hydroxyl forms hydrogen bond zinc-bound water molecule, causing further activation molecule reducing pK(a) value. pH dependency solvent isotope effects support proposition. A unified catalytic mechanism is proposed can account different behavior observed CPA-catalyzed peptide substrates.