Carboxypeptidase site-directed mutagenesis

U. H. Mortensen, S. J. Remington, K. Breddam, Site-Directed Mutagenesis on (Serine) Carboxypeptidase Y. A Hydrogen Bond Network Stabilizes the Transition State by Interaction with the C-Terminal Carboxylate Group of the Substrate , Biochemistry 1994, 33, 508-513. 

From crystallographic studies, fast reaction kinetics and site-directed mutagenesis to produce mutant enzymes differing from the native enzyme in one or more specified residues, the mechanism in (46) has been proposed for the attack of water at the carbonyl carbon of benzoylglycylphenylalanine bound to carboxypeptidase (Christianson and Lipscomb, 1989). 

Chiba, Y., Midorikawa, T., and Ichishima, E. (1995). Cloning and expression of the carboxypeptidase gene from Aspergillus saitoi and determination of the catalytic residues by site-directed mutagenesis. Biochem. J., 308, 405-409. 

Hypotheses regarding the consequences of specific amino acid replacements on substrate specificity can now be tested experimentally through site-directed mutagenesis of carboxypeptidase.25 Site-specific mutations can be introduced into cloned DNA by several highly efficient and simple methods. These methods have been summarized26 and are not detailed here. 

On the basis of chemical modification studies, Tyr 198 of carboxypeptidase A was proposed to act as a proton donor (i.e., a general acid) in the mechanism of catalysis. However, when Tyr 198 was replaced with Phe by means of site-directed mutagenesis, the modified enzyme retained substantial enzymatic activity, indicating that the tyrosyl hydroxyl may not have a specific role in catalysis. 

Gardell, S. J., Craik, C. S., Hilvert, D., Urdea, M. S. and Rutter, W. J. (1985) Site directed mutagenesis shows that tyrosine 248 of Carboxypeptidase A does not play a cmcial role in catalysis, Nature 317, 551-555. 

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