Mechanism-Based Studies of the Active Site-Directed Inhibition and Activation of Enzyme Transketolase

Document Type: Research Article

Author

Department of Biochemistry, School of Medicine, University of Tehran, Tehran, I.R. IRAN

Abstract

Derivatives of phenyl-keto butenoic acids have been reported to be inhibitors of pyruvate decarboxylase, (PDC). The inhibition of transketolase, a thiamine requiring enzyme such as PDF, by meta nitrophenyl derivative of 2-oxo-3-butenoic acid (MNPB) is reported here. These studies indicate that the inhibitor binds to the enzyme at the active site. A two-step inhibition was observed, first the inhibitor reacts with the enzyme on one site- non-cooperative with Mg2+, and TPP, inhibiting the enzyme, second in higher concentration of the inhibitor an abrupt enhancement of the inhibition takes place. In the absence of cofactors, the lower concentration of the inhibitor caused an enhancement of activation to 150% of original enzyme activity followed by a drop to a low 50%  in 60 minutes. Higher concentration of the inhibitor produced an inhibition with a half-life that was pronouncedly larger than when cofactors were present (). We conclude therefore that the enzyme contains a regulatory and a catalytic site with the regulatory site functional, without the aid of cofactors and that the cofactors are auxiliary for the action of the enzyme. The lowering effect of the reaction half-life by the cofactors is due to the rate enhancement caused by cofactors in the catalytic site of the enzyme.

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