Coenzyme binding in yeast pyruvate decarboxylase. Kinetic studies with thiamine diphosphate analogues.

Abstract

A kinetic investigation of the behavior of apopyruvate decarboxylase from Saccharomyces carlsbergensiswith thiamine pyrophosphate analogues gave the values of 23 μM as the Km for thiamine pyrophosphate and 20 μM as the Km for 2′‐ethylthiamine pyrophosphate. The Vfor the latter compound was 25%, that of thiamine pyrophosphate. Inhibitor constants, Ki, were determined for the following competitive inhibitors of thiamine pyrophosphate with the apoenzyme, (the values are for the pyrophosphate esters): tetrahydrothiamine, 6.5 μM; oxythiamine, 20 μM; 2′‐n‐butyIthiamine, 45 μM; 2′‐methoxythiamine, 70 μM; pyrithiamine, 78 μM; 2′‐demethylthiamine, 220 μM; 2′‐hydroxythiamine, 380 μM. None of the inhibitors exhibited coenzyme activity.A hydrophobic interaction of the 2′‐methyl group of thiamine pyrophosphate with the apoenzyme has been proposed and a model for the coenzyme binding site of pyruvate decarboxylase, consisting of a pyrophosphate and a pyrimidine binding region, is discussed.