Leu254 Residue and Calcium Ions as New Structural Determinants of Carboxypeptidase T Substrate Specificity

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A. M. Grishin, V. Kh. Akparov^*, and G. G. Chestukhina

Institute of Genetics and Selection of Industrial Microorganisms, 1-yi Dorozhnyi Proezd 1, 117545 Moscow, Russia; fax: (495) 315-0501; E-mail: vhar@ostrov.net

^* To whom correspondence should be addressed.

Received April 17, 2008; Revision received May 13, 2008

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New determinants of Thermoactinomyces vulgaris carboxypeptidase T (CPT) substrate specificity--structural calcium ions and Leu254 residue--were found by means of steady-state kinetics and site-directed mutagenesis. The removal of calcium ions shifted the selectivity profile of hydrolysis of tripeptide substrates with C-terminal Leu, Glu, and Arg from 64/1.7/1 to 162/1.3/1. Substitution of the hydrophobic Leu254 in CPT for polar Asn did not change hydrolysis efficiency of substrates with C-terminal Leu and Arg, but resulted in more than 28-fold decrease in activity towards the substrate with C-terminal Glu. It is shown that the His68 residue is not a structural determinant of CPT specificity.

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KEY WORDS: carboxypeptidase T, substrate specificity, rational redesign, protein engineering, site-directed mutagenesis

DOI: 10.1134/S0006297908100118

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