The role of Tyr248 probed by mutant bovine carboxypeptidase A: insight into the catalytic mechanism of carboxypeptidase A.
We have investigated the function of Tyr248 using bovine wild-type CPA and its Y248F and Y248A mutants to find that the K(M) values were increased by 4.5-11-fold and the k(cat) values were reduced by 4.5-10.7-fold by the replacement of Tyr248 with Phe for the hydrolysis of hippuryl-L-Phe (HPA) and N-[3-(2-furyl)acryloyl]-Phe-Phe (FAPP), respectively. In the case of O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate (ClCPL), an ester substrate, the K(M) value was increased by 2.5-fold, and the k(cat) was reduced by 20-fold. The replacement of Tyr248 with Ala decreased the k(cat) values by about 18- and 237-fold for HPA and ClCPL, respectively, demonstrating that the aromatic ring of Tyr248 plays a critical role in the enzymic reaction. The increases of the K(M) values were only 6- and 5-fold for HPA and ClCPL, respectively. Thus, the present study indicates clearly that Tyr248 plays an important role not only in the binding of substrate but also in the enzymic hydrolysis. The kinetic results may be rationalized by the proposition that the phenolic hydroxyl of Tyr248 forms a hydrogen bond with the zinc-bound water molecule, causing further activation of the water molecule by reducing its pK(a) value. The pH dependency study of k(cat) values and the solvent isotope effects also support the proposition. A unified catalytic mechanism is proposed that can account for the different kinetic behavior observed in the CPA-catalyzed hydrolysis of peptide and ester substrates.
Cho
J H
JH
Department of Life Sciences and Chemistry, Division of Molecular and Life, Pohang University of Science and Technology, San 31 Hyoja-dong, Pohang 790-784, Korea.
Kim
D H
DH
Lee
K J
KJ
Kim
D H
DH
Choi
K Y
KY
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry* / Q000378:metabolism*; D043422:Carboxypeptidases A; D002384:Catalysis; D002417:Cattle; D017931:DNA Primers; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine
D017931:DNA Primers; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; C049123:N-(hippuryl)phenylalanine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A