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Catalytic and specificity determinants in cis ‐3‐chloroacrylic acid dehalogenase: pre‐steady state kinetic analysis of active site loop mutants
Author(s) -
Schroeder Gottfried K,
Huddleston Jamison P,
Robertson Brooklyn A,
Johnson Kenneth A,
Whitman Christian P
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.lb222
Subject(s) - dehalogenase , chemistry , stereochemistry , active site , enzyme , proline , mutant , superfamily , stereospecificity , amino acid , residue (chemistry) , catalysis , biochemistry , receptor , gene
cis ‐3‐Chloroacrylic acid dehalogenase ( cis ‐CaaD) is a member of the tautomerase superfamily, with the characteristic α‐β ‐α fold and catalytic Pro‐1. Cg10062, a cis ‐CaaD homologue, shares 53% sequence similarity with cis ‐CaaD including six key active site residues. However, Cg10062 is a poor cis ‐CaaD: it has much lower catalytic efficiency and lacks stereospecificity. This dichotomy indicates that a fully functional cis ‐CaaD requires elements beyond the core set. Hence, a pre‐steady state kinetic analysis of cis ‐CaaD was performed to define further the characteristics of the tautomerase superfamily, including catalytic and specificity determinants. The results could be used to assign function to other cis ‐CaaD homologues. Replacement of a glutamine residue in a flexible loop region near the active site of cis ‐CaaD with proline (present in Cg10062) reduced the rate of each kinetically significant step of the catalytic cycle, based on a global fit of all steady and transient state kinetic data. This result holds important implications for the role of loop identity and movement in defining enzyme specificity and function. Supported by NIH GM 65324 and the Welch Foundation F‐1334 .