title: Use of directed mutagenesis to probe the role of tyrosine 198 in the catalytic mechanism of carboxypeptidase A.
abstract: Derivatization of Tyr198 in carboxypeptidase A (CPA) results in lowered catalytic activity toward peptide substrates (Cueni, L., and Riordan, J.F. (1978) Biochemistry 17, 1834-1842). We have synthesized via directed mutagenesis a rat CPA variant [Phe198] CPA containing a Tyr198-to-Phe substitution in order to test whether the phenolic hydroxyl plays a critical role in catalysis. A double mutant [Phe193, Phe248]CPA in which both Tyr198 and Tyr248 have been replaced by phenylalanine has also been engineered. Enzymatic characterization of [Phe198]CPA indicates that the Tyr198 hydroxyl is not obligatory for the hydrolysis of peptide and ester substrates. Furthermore, parallel studies with [Phe198, Phe248]CPA show that simultaneous removal of both the Tyr198 and Tyr248 hydroxyls does not abolish catalytic activity. Analysis of the acetylated derivatives of [Phe198]CPA, [Phe248]CPA, and [Phe198, Phe248]CPA establishes that Tyr198 and Tyr248 are the active site tyrosines which are modified by N-acetylimidazole. In addition, the perturbations of enzymatic activity which accompany acetylation of native CPA can be largely assigned to derivatization of Tyr248. The changes in the kinetic constants of substrate hydrolysis due to the Tyr198-to-Phe substitution are manifested as small decreases in the kcat values, but the Km values are essentially unaffected. This exclusive effect on the kcat values suggests that the Tyr198 hydroxyl participates in catalysis by stabilizing the rate-determining transition-state complex.
mesh_terms: D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000235:genetics* / Q000378:metabolism; D043422:Carboxypeptidases A; D014644:Genetic Variation; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014443:Tyrosine
chemical_list: D014443:Tyrosine; D002268:Carboxypeptidases; D043422:Carboxypeptidases A