Chemical modifications to study mutations that affect the ability of the general base (E268) to function in human...

The action of a general base is needed in two possible steps during the aldehyde dehydrogenase catalyzed oxidation of an aldehyde to an acid. The base is glutamate at position 268 in the cytosolic and mitochondrial class 1 and 2 enzyme. A chemical modification approach was undertaken to determine if the base were necessary in the initial attack of the nucleophilic cysteine (302) on the aldehyde as well as the attack by water on the acyl intermediate formed after the aldehyde is oxidized. A metabolite of disulfiram, S-methyl-N,N-diethylthiocarbamoyl sulfoxide (MeDTC-SO), was used as the modifying agent. Three recombinantly expressed mutant forms of the human mitochondrial enzyme along with the native one were used. These were the E268Q mutant that was lacking the general base; the E487K Oriental variant of the enzyme and R475Q, a mutant possessing the residue that binds to E487. As expected, the E268Q mutant was inactivated very slowly compared with the native or other mutants that were inactivated more slowly than the native enzyme. The presence of NAD did not increase the rate of inactivation except with the R475Q mutant. It is concluded that it is necessary to activate the cysteine at the active site to make it a good nucleophile as well to activate water during the hydrolysis of the thio-acyl intermediate. Further, it is surmised that the reason some mutants have a lowered specific activity is that in those the general base is not capable of functioning as it does in the native enzyme.