Partial Characterization of Pyruvate Decarboxylase Isolated From Germinating Pea Seeds

Abstract

Pyruvate decarboxylase (EC 4.1.1.1), isolated from 4-day-old germinating peas, was precipitated from a sodium phosphate extract when (NH4)2SO4 was increased from 15 to 30% saturation, desalted on Sephadex G-25 or by dialysis for 24 h, and then chromatographed on a DEAE-cellulose column. This procedure increased the specific activity of the enzyme 120-fold compared with the sodium phosphate extract. The behaviour of the enzyme during gel filtration indicates that it has a high molecular weight. The pea enzyme exhibits a sigmoid dependence on the pyruvate concentration; reaction velocity is half-maximal at a substrate concentration of 1.8 mM and the Hill coefficient is 1.8. Thiamin pyrophosphate (TPP) is the coenzyme, which is relatively firmly bound to the apoenzyme, but can be removed by dialysis for 48 h. The apoenzyme is activated optimally at 2 mM TPP and inhibited by concentrations above 4 mM. The pH optimum for pea pyruvate decarboxylase is 5.8 and maximal temperature stability occurs at 48°C.