INVOLVEMENT OF GLUTAMATE-268 IN THE ACTIVE-SITE OF HUMAN LIVER MITOCHONDRIAL (CLASS-2) ALDEHYDE DEHYDROGENASE AS PROBED BY SITE-DIRECTED MUTAGENESIS

On the basis of chemical modification studies, it was postulated that glutamate 268 was a component of the active site of liver aldehyde deh ydrogenase [Abriola, D.P., Fields, R., MacKerell, A.D., Jr., and Pietr uszko, R. (1987) Biochemistry 26, 5679-5684]. To study its role, the r esidue in human liver mitochondrial (class 2) aldehyde dehydrogenase w as mutated to an aspartate, a glutamine, or a lysine, and the enzyme w as expressed in Escherichia coli. The mutations did not affect the K-m values for NAD or propionaldehyde, but grossly affected the catalytic activity of the enzymes when compared to recombinantly expressed nati ve enzyme; the mutant enzymes had less that 0.4% of the specific activ ity of the recombinantly expressed native aldehyde dehydrogenase. The mutations also caused a long lag phase to occur prior to the steady st ate phase of the reaction. The activity of the mutant enzymes could no t be restored by the addition of general bases such as sodium acetate, sodium formate, or imidazole. The K-d for NADH was essentially identi cal for the E268Q mutant and native enzyme. The three mutant forms of the enzyme possessed less than 0.8% of the esterolytic activity of the recombinantly expressed native enzyme. Pre-steady state analysis show ed that there was no burst of NADH formation in the dehydrogenase reac tion or of p-nitrophenol formation in the esterase reaction. This can be interpreted as implying that glutamate 268 may function as a genera l base necessary for the initial activation of the essential cysteine residue (302), rather than being involved in only the deacylation or h ydride transfer step. Alternatively, glutamate 268 could function as a component of a charge relay triad necessary to activate the nucleophi lic residue. Furthermore, it appears that esterase and dehydrogenase r equire the same active site components, for both the dehydrogenase act ivity and esterase activity were essentially abolished when glutamate 268 was changed to another residue.