Abstract
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 ba...Continue Reading
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