Mechanism of r : econstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium.

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Title:	Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium.
Author:	Vaccaro, J A : Crane, E J : Harris, T K : Washabaugh, M W
Citation:	Biochemistry. 1995 Oct 3; 34(39): 12636-44
Abstract:	Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and alpha 4 isozyme (alpha 4-PDC) exhibits biphasic kinetics with 52 +/- 11% of the PDC reacting with k1 = (1.0 +/- 0.3) x 10(-2) s-1 and 48 +/- 12% of the PDC reacting with k2 = (1.1 +/- 0.6) x 10(-1) s-1 when TDP (KTDP = 0.5 +/- 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 +/- 0.5) x 10(-2) s-1 upon addition of Mg2+ (KMg2+ = 0.2 +/- 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 less than or = 3-fold and has no effect on the value of k1.(ABSTRACT TRUNCATED AT 250 WORDS)
Review References:	None
Notes:	None
Language:	English
Publication Type:	Journal-Article
Keywords:	Isoenzymes chemistry : Magnesium chemistry : Pyruvate Decarboxylase chemistry : Saccharomyces cerevisiae enzymology : Thiamine Pyrophosphate chemistry
URL:	http://pubs.acs.org/journals/bichaw/index.html