Significant Catalytic Roles for Glu47 and Gln 110 in All Four of the C−C Bond-Making and -Breaking Steps of the Reactions of Acetohydroxyacid Synthase II

摘要

Acetohydroxy acid synthase (AHAS) is a thiaminndiphosphate (ThDP)-dependent enzyme that catalyzes the firstncommon step in the biosynthesis of branched-chain aminonacids, condensation of pyruvate with a second 2-ketoacid tonform either acetolactate or acetohydroxybutyrate. AHAS iso-nzyme II from Escherichia coli is specific for pyruvate as the firstndonor substrate but exhibits a 60-fold higher specificity forn2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate.nIn previous studies relying on steady state and transient kinetics, substrate competition and detailed analysis of the distribution ofnintermediates in the steady-state, we have identified several residues which confer specificity for the donor and acceptor substrates,nrespectively. Here, we examine the roles of active site polar residues Glu47, Gln110, Lys159, and His251 for elementary steps ofncatalysis using similar approaches. While Glu47, the conserved essential glutamate conserved in all ThDP-dependent enzymesnwhose carboxylate is in H-bonding distance of the ThDP iminopyrimidine N10n, is involved as expected in cofactor activation,nsubstrate binding, and product elimination, our studies further suggest a crucial catalytic role for it in the carboligation of thenacceptor and the hydroxyethyl-ThDP enamine intermediate. The Glu47-cofactor proton shuttle acts in concert with Gln110 in thencarboligation. We suggest that either the transient oxyanion on the acceptor carbonyl is stabilized by H-bonding to the glutaminenside chain, or carboligation involves glutamine tautomerization and the elementary reactions of addition and protonation occur in anconcertedmanner. This is in contrast to the situation in other ThDP enzymes that catalyze a carboligation, such as, e.g., transketolasenor benzaldehyde lyase, where histidines act as general acid/base catalysts. Our studies further suggest global catalytic roles fornGln110 and Glu47, which are engaged in allmajor bond-breaking and bond-making steps. In contrast to earlier suggestions, Lys159nhas a minor effect on the kinetics and specificity of AHAS II, far less than does Arg276, previously shown to influence the specificitynfor a 2-ketoacid as a second substrate. His251 has a large effect on donor substrate binding, but this effect masks any other effects ofnreplacement of His251.