THE OXYGENASE REACTION OF ACETOLACTATE SYNTHASE

In addition to the physiological reactions catalyzed by acetolactate s ynthase, it supports an oxygen-consuming side reaction. Although the s ynthase and oxygenase activities are activated to somewhat different e xtents by various metals (Mn2+, Mg2+, Ca2+, Co2+, Zn2+, Ni2+, Cd2+, Cu 2+, Ba2+, Al3+), the modest degree of these differences (at most 6-fol d) and the high degree of promiscuity of the enzyme with respect to it s metal requirement suggest that the metal is not intimately involved in the chemistry of either reaction. Saturation of the oxygenase react ion occurs at pyruvate concentrations below the limit of sensitivity f or the oxygen electrode (<10 muM), at higher concentrations pyruvate i nhibits the rate of oxygen consumption. At a noninhibitory concentrati on of pyruvate (1 mM), inhibition of the reaction is also observed wit h alpha-ketobutyrate. Inhibition of the oxygenase reaction by high con centrations of pyruvate or alpha-ketobutyrate is presumably due to com petition between these substrates and molecular oxygen for a common ca rbanionic reaction intermediate, the conjugate base of (hydroxyethyl)t hiamin pyrophosphate. Inhibition of the reaction indicates that the la ctylthiamin pyrophosphate intermediate can decarboxylate prior to bind ing of the second pyruvate or alpha-ketobutyrate. At high concentratio ns of pyruvate or alpha-ketobutyrate, only incomplete inhibition of th e oxygenase reaction is achieved (65-89 % or 89-93 % maximal inhibitio n, respectively). This incomplete inhibition of the oxygenase reaction by alpha-keto acids indicates that the reaction is not Theorell-Chanc e with respect to addition of the second alpha-keto acid and that oxyg en has more than one route of access to the carbanionic reaction inter mediate.