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High Resolution Crystal Structures of the Acetohydroxyacid Synthase-Pyruvate Complex Provide New Insights into Its Catalytic Mechanism

Abstract:

Acetohydroxyacid synthase (AHAS) is the first enzyme in the biosynthesis pathway of the branched-chain amino acids, catalyzing the condensation of pyruvate with another molecule of pyruvate or with 2-ketobutyrate, to produce 2-acetolactate or 2-acetohydroxybutyrate, respectively. The catalytic subunit of the dimeric enzyme has thiamin diphosphate (ThDP), a divalent metal ion, flavin adenine dinucleotide (FAD), and two molecules of oxygen (O2(I) and O2(II)) as cofactors. Here, crystal structures of Saccharomyces cerevisiae AHAS in complex with pyruvate provide novel insights into the mechanistic features of this enzyme including: i) The precise position taken by pyruvate molecules as they enter the active site (i. e. prior to catalysis occurring) with conformations suitable for the transfer of electrons to/from O2(I) and FAD; ii) The formation of ternary donor-acceptor-O2(I) complexes and iii) The location of O2(II) relative to the substrate showing that it plays a critical role in the organization of substrate for catalysis. These structural data, accompanied by electron paramagnetic resonance evidence that a radical is produced during AHAS catalysis, lead to the proposal that FAD and O2are involved in an indirect one-electron redox cycle. In this mechanism, the spatial configurations of O2and FAD in the active site can allow electrons to be exchanged with the substrates and catalytic intermediates to satisfy and control the overall AHAS catalyzed reaction.