CRITICAL GLUTAMIC-ACID RESIDUES AFFECTING THE MECHANISM AND NUCLEOTIDE SPECIFICITY OF VIBRIO-HARVEYI ALDEHYDE DEHYDROGENASE

Fatty aldehyde dehydrogenase (ALDH) from the luminescent marine bacter ium, Vibrio harveyi, differs from other ALDHs in its unique specificit y and high affinity for NADP(+). Two glutamic acid residues, Glu253 an d Glu377, which are highly conserved in ALDHs, were investigated in th e present study. Mutation or Glu253 to Ala decreased the k(cat) for AL DH activity by over four orders of magnitude without a significant cha nge in the K-m values for substrates or the ability to interact with n ucleotides. Both thioesterase activity and a pre-steady-stale burst of NAD(P)N were also eliminated, implicating Glu253 in promoting the nuc leophilicity of the cysteine residue(Cys289) involved in forming the t hiohemiacetal intermediate in the enzyme mechanism. Mutation of Glu377 to Gln (E377Q mutant) selectively decreased the k(cat) for NAD(+)-dep endent ALDH activity (>10(2)-fold) compared to only a 6-fold loss in N ADP(+)-dependent activity without comparable changes to the K-m values for substrates. Consequently, the E377Q mutant had a very high specif icity for NADP(+)(k(cat)/K-m > 10(3) of that for NAD(+)) which was ove r 20 times higher than that or the wild-type ALDH. Although a pre-stea dy-state burst of NAD(P)H was eliminated by this mutation, thioesteras e activity was completely retained, Using [1-H-3]acetaldehyde as a sub strate, a significant deuterium isotope effects was observed, implicat ing Glu377 in the hydride transfer step and not in acylation or releas e of the acyl group from the cysteine nucleophile. The increase in spe cificity of the E377Q mutant for NADP(+) is consistent with a change i n the rate-limiting step determining k(cat) from nucleotide-dependent NAD(P)H dissociation to hydride transfer. The results provide biochemi cal evidence that the two highly conserved Glu residues are involved i n different functions in the active site of V. harveyi ALDH.