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Biochemistry. 2006 Nov 14;45(45):13517-27.

Function of a conserved loop of the beta-domain, not involved in thiamin diphosphate binding, in catalysis and substrate activation in yeast pyruvate decarboxylase.

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  • 1Department of Chemistry, Rutgers, the State University of New Jersey, Newark, New Jersey 07102, USA.

Abstract

The X-ray crystal structure of pyruvamide-activated yeast pyruvate decarboxylase (YPDC) revealed a flexible loop spanning residues 290 to 304 on the beta-domain of the enzyme, not seen in the absence of pyruvamide, a substrate activator surrogate. Site-directed mutagenesis studies revealed that residues on the loop affect the activity, with some residues reducing k(cat)/K(m) by at least 1000-fold. In the pyruvamide-activated form, the loop located on the beta domain can transfer information to the active center thiamin diphosphate (ThDP) located at the interface of the alpha and gamma domains. The sigmoidal v(0)-[S] curve with wild-type YPDC attributed to substrate activation is modulated for most variants, but is not abolished. Pre-steady-state stopped-flow studies for product formation on these loop variants provided evidence for three enzyme conformations connected by two transitions, as already noted for the wild-type YPDC at pH 5.0 [Sergienko, E. A., and Jordan, F. (2002) Biochemistry 41, 3952-3967]. (1)H NMR analysis of the intermediate distribution resulting from acid quench [Tittmann et al. (2003) Biochemistry 42, 7885-7891] with all YPDC variants indicated that product release is rate limiting in the steady state. Apparently, the loop is not solely responsible for the substrate activation behavior, rather it may affect the behavior of residue C221 identified as the trigger for substrate activation. The most important function of the loop is to control the conformational equilibrium between the "open" and "closed" conformations of the enzyme identified in the pyruvamide-activated structure [Lu et al. (2000) Eur. J. Biochem. 267, 861-868].

PMID:
17087505
DOI:
10.1021/bi0615588
[PubMed - indexed for MEDLINE]
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