Absolute configuration of the product of the acetolactate synthase reaction by a novel method of analysis using acetolactate decarboxylase

Abstract

Pyruvate and 2-oxobutanoate were incubated with acetolactate synthase [acetolactate pyruvate lyase (carboxylating, EC 4.1.3.18)] in the presence of acetolactate decarboxylase [(S)-2-hydroxy-2-methyl-3-oxobutanoate carboxy-lyase, EC 4.1.1.5)]. The exclusive production of 3-hydroxypentan-2-one showed that the α-acetohydroxybutyrate (2-ethyl-2-hydroxy-3-oxobutanoate) produced by acetolactate synthase had the (S)-configuration. The ability of acetolactate decarboxylase to catalyse rearrangement of the (R)-enantiomer of substrates via carboxylate migration was investigated using [3,4-¹³C₂]-α-acetolactate, in which the degeneracy of the rearrangement of the normal substrate was broken by isotopic labelling. The predicted sequential formation of [1,2-¹³C₂]- and [3,4-¹³C₂]-acetoin was observed.