Homoserine dehydrogenase from Saccharomyces cerevisiae: kinetic mechanism and stereochemistry of hydride transfer

Homoserine dehydrogenase (HSD), which is required for the synthesis of threonine, isoleucine and methionine in fungi, is a potential target for novel antifungal drugs. In order to design effective inhibitors, the kinetic mechanism of Saccharomyces cerevisiae HSD and the stereochemistry of hydride transfer were examined. Product inhibition experiments revealed that yeast HSD follows an ordered Bi Bi kinetic mechanism, where NAD(P)H must bind the enzyme prior to aspartate semialdehyde (ASA) and homoserine is released first followed by NAD(P)+. H-(1,2,4-triazol-3-yl)-D,L-alanine was an uncompetitive inhibitor of HSD with respect to NADPH (K(ii)=3.04+/-0.18 mM) and a noncompetitive inhibitor with respect to ASA (K(is)=1.64+/-0.36 mM, K(ii)=3.84+/-0.46 mM), in agreement with the proposed substrate order. Both kinetic isotope and viscosity experiments provided evidence for a very rapid catalytic step and suggest nicotinamide release to be primarily rate limiting. Incubation of HSD with stereospecifically deuterated NADP[2H] and subsaturating amounts of aspartate semialdehyde revealed that the pro-S NADPH hydride is transferred to the aldehyde. The pH dependence of steady state kinetic parameters indicate that ionizable groups with basic pKs may be involved in substrate binding, consistent with the observation of Lys223 at the enzyme active site in the recently determined 3D structure [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. These findings provide the requisite foundation for future exploitation of fungal HSD in inhibitor design.