ALDH5 (aka succinic semialdehyde dehydrogenase) is a NAD(+)-dependent aldehyde dehydrogenase crucial for the proper removal of the GABA metabolite succinic semialdehyde (SSA). All known ALDH5 family members contain the conserved amino acid sequence "MITRK". Our studies of rat ALDH5A indicate that residue R166 in this sequence may play a role in the substrate specificity of ALDH5A for the gamma-carboxylated succinic semialdehyde versus other aliphatic and aromatic aldehydes including acetaldehyde and benzaldehyde. We tested the hypothesis that the R166 residue regulates aldehyde specificity by utilizing rat ALDH5A wild-type (R166wt) and R166K, R166H, R166A, and R166E mutants. The V(MAX) using SSA fell whereas the K(M) for SSA increased for all mutants analyzed yielding k(cat)/K(M) (s(-1)/microM) ratios of 52.3 (R166wt), 5.5 (R166K), 0.01 (R166H), 0.008 (R166E), and 0.004 (R166A). Utilization of acetaldehyde by the R166H mutant was similar to R166wt with k(cat)/K(M)'s of 0.003 and 0.002, respectively. Almost no activity towards acetaldehyde was noted for the R166E and R166A mutants. Unexpectedly, the K(M) for NAD(+) changed: 21 microM (R166wt), 81 microM (R166K), 63 microM (R166H), 35 microM (R166E) and 44 microM (R166A). As release of NADH can be a rate-limiting step for ALDH activity, NADH binding was evaluated for R166wt and R166H enzymes. The K(D) of NADH for R166H (0.9 microM) was 11-fold less than that of ALDH5A wt (10.3 microM) and possibly explains the increase in the K(M) for NAD(+). Furthermore, data using R166K and R166H mutants demonstrate that inhibition of enzyme activity by low pH is regulated in part by the R166 residue. Our data indicate that the R166 residue of ALDH5A regulates multiple enzymatic functions.