Inhibition of Recombinant Human Mitochondrial and Cytosolic Aldehyde Dehydrogenases by Two Candidates for the Active Metabolites of Disulfiram
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- 作者：Jennifer P. Lam ; Dennis C. Mays ; and James J. Lipsky
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：November 4, 1997
- 年：1997
- 卷：36
- 期：44
- 页码：13748 - 13754
- 全文大小：164K
- 年卷期：v.36,no.44(November 4, 1997)
- ISSN：1520-4995
文摘
We expressed recombinant human cytosolic (ALDH1, highKm) and mitochondrial aldehydedehydrogenase (ALDH2, low Km) inEscherichia coli and purified the enzymes to homogeneity toexaminethe nature of inhibition of human ALDH by disulfiram, its confirmedmetabolite S-methyl N,N-diethylthiocarbamate (MeDTC) sulfoxide, and its proposed metaboliteMeDTC sulfone. Disulfiram,MeDTC sulfoxide, and MeDTC sulfone, respectively, were potentinhibitors with IC50 values of 0.15 ±0.02 
M, 0.27 ± 0.04 M, and 0.12 ± 0.02 M for ALDH1, and1.45 ± 0.40 M, 1.16 ± 0.56, and 0.40± 0.10 M for ALDH2. Extensive dialysis did not restore theactivity of the inactivated enzyme, indicatingirreversible inhibition. Both the esterase and dehydrogenaseactivities of ALDH2 were inhibited to thesame extent by MeDTC sulfone and sulfoxide, suggesting that bothcatalytic sites are closely linked.The time course of inhibition of ALDH appeared to be first-orderfor both MeDTC sulfone and MeDTCsulfoxide. Kitz and Wilson plots of the half-life of inactivationversus 1/[inhibitor] indicated that thereactions between ALDH and inhibitors were bimolecular. Thepseudobimolecular rate constants(k3/KI)for the ALDH-inhibitor reactions were 1 × 105, 1 ×104, 3 × 103, and 1 × 103s-1 M-1ALDH1-sulfone, ALDH1-sulfoxide, ALDH2-sulfone, and ALDH2-sulfoxide,respectively. ALDH2 was notsignificantly protected from inactivation from either MeDTC sulfoxideor MeDTC sulfone by NAD alone,but high concentrations of NAD and acetaldehyde completely preventedinhibition. Since disulfiram israpidly metabolized in vivo, it is believed that disulfiramis too short-lived to inhibit ALDH directly.The results of our study indicate that MeDTC sulfoxide and sulfoneare potent inhibitors of human ALDHand are reasonable candidates for the proximal inhibitors of ALDHfollowing disulfiram administration.
M, 0.27 ± 0.04 M, and 0.12 ± 0.02 M for ALDH1, and1.45 ± 0.40 M, 1.16 ± 0.56, and 0.40± 0.10 M for ALDH2. Extensive dialysis did not restore theactivity of the inactivated enzyme, indicatingirreversible inhibition. Both the esterase and dehydrogenaseactivities of ALDH2 were inhibited to thesame extent by MeDTC sulfone and sulfoxide, suggesting that bothcatalytic sites are closely linked.The time course of inhibition of ALDH appeared to be first-orderfor both MeDTC sulfone and MeDTCsulfoxide. Kitz and Wilson plots of the half-life of inactivationversus 1/[inhibitor] indicated that thereactions between ALDH and inhibitors were bimolecular. Thepseudobimolecular rate constants(k3/KI)for the ALDH-inhibitor reactions were 1 × 105, 1 ×104, 3 × 103, and 1 × 103s-1 M-1ALDH1-sulfone, ALDH1-sulfoxide, ALDH2-sulfone, and ALDH2-sulfoxide,respectively. ALDH2 was notsignificantly protected from inactivation from either MeDTC sulfoxideor MeDTC sulfone by NAD alone,but high concentrations of NAD and acetaldehyde completely preventedinhibition. Since disulfiram israpidly metabolized in vivo, it is believed that disulfiramis too short-lived to inhibit ALDH directly.The results of our study indicate that MeDTC sulfoxide and sulfoneare potent inhibitors of human ALDHand are reasonable candidates for the proximal inhibitors of ALDHfollowing disulfiram administration.