Coenzyme Binding in Yeast Pyruvate Decarboxylase

A kinetic investigation of the behavior of apopyruvate decarboxylase from Saccharomyces carlsbergensis with thiamine pyrophosphate analogues gave the values of 23 μM as the K m for thiamine pyrophosphate and 20 μM as the K m for 2′‐ethylthiamine pyrophosphate. The V for the latter compound was 25%, that of thiamine pyrophosphate. Inhibitor constants, K i , 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.