Revised Manuscript Received October 11, 1994® abstract: On the basis of chemical modification studies, it was postulated that glutamate 268 was a component of the active site of liver aldehyde dehydrogenase [Abriola, D. P., Fields, R., MacKerell, A. D., Jr., & Pietruszko, R.(1987) Biochemistry 26, 5679—5684], To study its role, the residue in human liver mitochondrial (class 2) aldehyde dehydrogenase was mutated to an aspartate, a glutamine, or a lysine, and the enzyme was expressed in Escherichia coli. The mutationsdid not affect the Km values for NAD or propionaldehyde, but grossly affected the catalytic activity of the enzymes when compared to recombinantly expressed native enzyme; the mutant enzymes had less that 0.4% of the specific activity of the recombinantly expressed native aldehyde dehydrogenase. The mutations also caused a long lag phase to occur prior to the steady state phase of the reaction. The activity of the mutant enzymes could not be restored by the addition of general bases such as sodium acetate, sodium formate, or imidazole. The Kd for NADH was essentially identical for the E268Q mutant and native enzyme. The three mutant forms of the enzyme possessed less than 0.8% of the esterolytic activityof the recombinantly expressed native enzyme. Pre-steady state analysis showed that there was no burst of NADH formationin the dehydrogenase reaction or of p-nitrophenol formation in the esterase reaction. This can be interpreted as implying that glutamate 268 may function as a general base necessary for the initial activation of the essential cysteine residue (302), rather than being involved in only the deacylation or hydride transfer step. Alternatively, glutamate 268 could function as a component of a charge relaytriad necessary to activate the nucleophilic residue. Furthermore, it appears that esterase and dehydrogenase require the same active site components, for both the dehydrogenase activity and esterase activity were essentially abolished when glutamate 268 was changed to another residue.

In spite of the fact that mammalian aldehyde dehydroge-nase (ALDH) 1 was first purified to homogeneityin the early 1970s, all of the components of the active site have not been unequivocally identified. The enzymeappears to follow a mechanism similar to that catalyzed by glyceraldehyde-3-phosphate dehydrogenase (Feldman & Weiner, 1972; Wein-er, 1979). That is, an active site nucleophile attacks the aldehyde to form a thiohemiacetal intermediate, which is then oxidized to form an acyl enzyme. Site-directed mutagenesis