CATALYTIC POWER OF PYRUVATE DECARBOXYLASE - RATE-LIMITING EVENTS AND MICROSCOPIC RATE CONSTANTS FROM PRIMARY CARBON AND SECONDARY HYDROGEN ISOTOPE EFFECTS

被引:73
作者
ALVAREZ, FJ
ERMER, J
HUBNER, G
SCHELLENBERGER, A
SCHOWEN, RL
机构
[1] UNIV KANSAS,DEPT CHEM,LAWRENCE,KS 66045
[2] UNIV KANSAS,DEPT BIOCHEM,LAWRENCE,KS 66045
[3] MARTIN LUTHER UNIV,INST BIOCHEM,O-4020 HALLE,GERMANY
关键词
D O I
10.1021/ja00022a030
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Isotope effects ([rate constant for light isotopic substrate]/[rate constant for heavy isotopic substrate]) for the action of the thiamin diphosphate dependent pyruvate decarboxylase of Saccharomyces carlsbergensis (EC 4.1.1.1) on pyruvate, pyruvate-1-C-13, pyruvate-2-C-13, and pyruvate-3-d3 have been determined for each of the steady-state kinetic parameters k/A (second-order in pyruvate), k/B (first-order in pyruvate), and k (zero-order in pyruvate). The 1-C-13 effects are 1.008 +/- 0.010 (k/A), 1.013 +/- 0.024 (k/B), and 1.024 +/- 0.006 (k). The 2-C-13 effects are 1.013 +/- 0.009 (k/A), 0.951 +/- 0.020 (k/B), and 1.039 +/- 0.004 (k). The 3-d3 effects are 0.883 +/- 0.013 (k/A), 0.881 +/- 0.026 (k/B), and 1.057 +/- 0.005 (k). Effects with 2-oxobutanoate and 2-oxobutanoate-3-d2 are 0.951 +/- 0.012 (k/A), 0.821 +/- 0.096 (k/B), and 1.057 +/- 0.005 (k). Pyruvate decarboxylase was already known to be hysteretically activated by the substrate, with pyruvate binding to the regulatory site with dissociation constant 8 mM and producing unimolecular activation (0.46 s-1) and deactivation (0.033 s-1). The isotope effects lead to rate constants for substrate binding to the catalytic site of 8.2 x 10(4) M-1 s-1, for substrate departure from the catalytic site of 120 s-1, for decarboxylation of 640 s-1, and for product release of 640 s-1. Pyruvate decarboxylase increases the rate of decarboxylation of pyruvate by thiamin alone by a factor of 3 x 10(12) at pH 6.2, 30-degrees-C. Under these conditions, conversion of activated enzyme and pyruvate to the enzymic species preceding decarboxylation is 4 x 10(12) times faster than the specific-base-catalyzed addition of thiamin to pyruvate. The enzymic species preceding decarboxylation reverts to activated enzyme and free pyruvate 6 x 10(9) times faster than the specific-base-catalyzed reversion of the adduct of thiamin and pyruvate to thiamin and free pyruvate. Enzymic decarboxylation is 10(7) times faster than decarboxylation of the adduct of thiamin and pyruvate.
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页码:8402 / 8409
页数:8
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