INVOLVEMENT OF GLUTAMATE-399 AND LYSINE-192 IN THE MECHANISM OF HUMANLIVER MITOCHONDRIAL ALDEHYDE DEHYDROGENASE

Mutation to the conserved Glu(399) Or Lys(192) caused the rate-limitin g step of human liver mitochondrial aldehyde dehydrogenase (ALDH2) to change from deacylation to hydride transfer (Sheikh, S., Ni, L., Hurle y, T, D,, and Weiner, H. (1997) J. Biol. Chem. 272, 18817-18822), Here we further investigated the role of these two NAD(+)-ribose-binding r esidues. The E399Q/K/H/D and K192Q mutants had lower dehydrogenase act ivity when compared with the native enzyme, No pre-steady state burst of NADH formation was found with the E399Q/K and R192Q enzymes when pr opionaldehyde was used as the substrate; furthermore, each mutant oxid ized chloroacetaldehyde slower than propionaldehyde, and a primary iso tope effect was observed for each mutant when [H-2]acetaldehyde was us ed as a substrate, However, no isotope effect was observed for each mu tant when alpha-[H-2]benzaldehyde was the substrate, A pre-steady stat e burst of NADH formation was observed for the E399Q/H and R192Q mutan ts with benzaldehyde, and p-nitrobenzaldehyde was oxidized faster than benzaldehyde. Hence, when aromatic aldehydes were used as substrates, the rate-limiting step remained deacylation for all these mutants, Th e rate-limiting step remained deacylation for the E399H/D mutants when either aliphatic or aromatic aldehydes were used as substrates, The K 192Q mutant displayed a change in substrate specificity, with aromatic aldehydes becoming better substrates than aliphatic aldehydes.