Catalytic power of pyruvate decarboxylase. Rate-limiting events and microscopic rate constants from primary carbon and secondary hydrogen isotope effects

Abstract

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 .I. 1) on pyruvate, pyruvate-]-% pyruvate-2-I3C, 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 l-"C effects are 1.008 f 0.010 ( k / A ) , 1.013 f 0.024 ( k / B ) , and 1.024 f 0.006 ( k ) . The 2-I3C effects are 1.013 f 0.009 ( k / A ) , 0.951 f 0.020 ( k / B ) , and 1.039 f 0.004 ( k ) . The 3-d3 effects are 0.883 f 0.013 ( k / A ) , 0.881 f 0.026 ( k / B ) , and 1.057 f 0.005 ( k ) . Effects with 2-oxobutanoate and 2-oxobutanoate-3-d2 are 0.951 f 0.012 ( k / A ) , 0.821 f 0.096 ( k / B ) , and 1.057 f 0.005 (k). Pyruvatedecarboxylase 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-l) and deactivation (0.033 s-l). The isotope effects lead to rate constants for substrate binding to the catalytic site of 8.2 X lo4 M-I s-l, for substrate departure from the catalytic site of 120 s-l, for decarboxylation of 640 s-l, and for product release of 640 s-l. Pyruvate decarboxylase increases the rate of decarboxylation of pyruvate by thiamin alone by a factor of 3 X 10l2 at pH 6.2, 30 OC. Under these conditions, conversion of activated enzyme and pyruvate to the enzymic species preceding decarboxylation is 4 X lo1* 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 IO9 times faster than the specific-base-catalyzed reversion of the adduct of thiamin and pyruvate to thiamin and free pyruvate. Enzymic decarboxylation is lo7 times faster than decarboxylation of the adduct of thiamin and pyruvate.

Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex

“…1)] mediated via loop motion has been proposed (3,4,27), the effect of disruption of this interaction is modest [H407A and E401K reduce k cat (E1ec specific) by Ϸ10-fold (4,27)]. Further, it is difficult to model an E1ec reference reaction, because there simply is no reaction in the absence of ThDP, whereas the protein supplies a 10 12 -fold rate acceleration over and above that by ThDP itself (28). Our results and conclusions are consistent with the generalized hypothesis in ref.…”

Lessons from thiamin-watching

“…The magnitude of the isotope effect could then be compared to that of the enzymic reaction in the elegant work of Schowen and his collaborators (ref. 12,13). They have been able to provide a detailed picture of the energetics of pyruvate decarboxylase catalysis from analysis of enzymic and nonenzymic isotope effects on each step.…”

“…Kim carefully studied the reaction by direct measurement using uv spectroscopy. Kim found that under the conditions where general base catalysis was observed lo undergoes an apparent internal redox reaction in which the C2a position of the hydroxybenzyl sidechain is oxidized to give a phenyl thiazole ketone (24 1 -hydroxybenzyl)-5-(2-hydroxyethyl)-4-methylthiazoley (13) while the C-N bond to the thiazolium nitrogen is reductively cleaved to give the 2,5-dimethyl-4-amino-1,3-pyrimidine (12). …”

Synthesis and 3D-QSARs Analyses of Herbicidal O,O-Dialkyl-1-phenoxyacetoxy-1-methylphosphonate Analogues as a New Class of Potent Inhibitors of Pyruvate Dehydrogenase

“…1.2.4.1) is the constituent structure that first affects the production of Acetyl-CoA using PDHc's TPP enzyme and cofactor's Mg 2+ metal as opposed to the other various procedures. 13,14 Therefore PDHc Elenzyme is a target of the pesticide design and the research was planned for the purpose of inhibiting this enzyme. 15 For the past few years, it was observed that phenoxyacetoxyalkylphosphonates derivatives showed high herbicidal activity as a PDH inhibitor.…”