INHIBITION OF RECOMBINANT HUMAN MITOCHONDRIAL AND CYTOSOLIC ALDEHYDE DEHYDROGENASES BY 2 CANDIDATES FOR THE ACTIVE METABOLITES OF DISULFIRAM

We expressed recombinant human cytosolic (ALDH1, high K-m) and mitocho ndrial aldehyde dehydrogenase (ALDH2, low K-m) in Escherichia coli and purified the enzymes to homogeneity to examine the nature of inhibiti on of human ALDH by disulfiram, its confirmed metabolite S-methyl N,N- diethylthiocarbamate (MeDTC) sulfoxide, and its proposed metabolite Me DTC sulfone. Disulfiram, MeDTC sulfoxide, and MeDTC sulfone, respectiv ely, were potent inhibitors with IC50 values of 0.15 +/- 0.02 mu M, 0. 27 +/- 0.04 mu M, and 0.12 +/- 0.02 mu M for ALDH1, and 1.45 +/- 0.40 mu M, 1.16 +/- 0.56, and 0.40 +/- 0.10 mu M for ALDH2. Extensive dialy sis did not restore the activity of the inactivated enzyme, indicating irreversible inhibition. Both the esterase and dehydrogenase activiti es of ALDH2 were inhibited to the same extent by MeDTC sulfone and sul foxide, suggesting that both catalytic sites are closely linked. The t ime course of inhibition of ALDH appeared to be first-order for both M eDTC sulfone and MeDTC sulfoxide. Kitz and Wilson plots of the half-li fe of inactivation versus 1/[inhibitor] indicated that the reactions b etween ALDH and inhibitors were bimolecular. The pseudobimolecular rat e constants (k(3)/K-I) for the ALDH-inhibitor reactions were 1 x 10(5) , 1 x 10(4), 3 x 10(3), and 1 x 10(3) s(-1) M-1 ALDH1-sulfone, ALDH1-s ulfoxide, ALDH2-sulfone, and ALDH2-sulfoxide, respectively. ALDH2 was not significantly protected from inactivation from either MeDTC sulfox ide or MeDTC sulfone by NAD alone, but high concentrations of NAD and acetaldehyde completely prevented inhibition. Since disulfiram is rapi dly metabolized in vivo, it is believed that disulfiram is too short-l ived to inhibit ALDH directly. The results of our study indicate that MeDTC sulfoxide and sulfone are patent inhibitors of human ALDH and ar e reasonable candidates for the proximal inhibitors of ALDH following disulfiram administration.