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PEDS Advance Access published online on May 30, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn031
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Structural principles of the broad substrate specificity of Thermoactinomyces vulgaris carboxypeptidase T—role of amino acid residues at positions 260 and 262

A.M. Grishin, V. Kh Akparov1 and G.G. Chestukhina

The V.M. Stepanov Laboratory of Protein Chemistry, Institute of Genetics and Selection of Industrial Microorganisms 1, 1-ii Dorozhnyi Proezd, 117545 Moscow, Russian Federation

1 To whom correspondence should be addressed. E-mail: vhar{at}ostrov.net

An influence of residues at positions 260 and 262 on a broad substrate specificity of Thermoactinomyces vulgaris carboxypeptidase T (CPT) has been studied by means of site-directed mutagenesis. The structure of the S1'-site of CPT is similar to those of pancreatic carboxypeptidases A (CPA) and B (CPB); however, the enzyme is capable of cleaving off C-terminal hydrophobic (like CPA), C-terminal positively charged (like CPB), and negatively charged residues. The spatial alteration of the S1' site hydrophobic area in CPT by an insertion of one residue in the active site loop with Tyr255 by analogy with CPA and CPB did not change the enzyme specificity. The introduction of Ile262 (CPT D260G/T262I) led to a statistically significant reduction in activity towards charged substrates. The removal of a negative (CPT D260G) and placement of a positive charge (CPT D260G/T262K and CPT D260G/T262R) in the S1' site shifted the specificity of the variants towards substrates with C-terminal Glu. The selectivity profile was 64:1.7:1 for wild-type CPT, 815:115:1 for CPT D260G, 3270:1060:1 for CPT D260G/T262K and 1:2.4:0 for CPT D260G/T262R for substrates with C-terminal Leu, Glu and Arg, respectively. The obtained results confirm the important role of the amino acid residues at positions 260 and 262 in determination of the CPT substrate specificity.

Keywords: carboxypeptidase T/protein engineering/rational redesign/site-directed mutagenesis/substrate specificity

Received January 15, 2008; revised April 30, 2008; accepted May 6, 2008.


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