MacKerell A D, MacWright R S, Pietruszko R
Biochemistry. 1986 Sep 9;25(18):5182-9. doi: 10.1021/bi00366a030.
Human liver aldehyde dehydrogenase isozymes E1 and E2 (EC 1.2.1.3) are both completely and irreversibly inactivated by bromoacetophenone (2-bromo-1-phenylethanone). Steady-state kinetics with both acetophenone and chloroacetophenone indicated interaction with the same enzyme form as the aldehyde substrate. Saturation kinetics with chloroacetophenone and bromoacetophenone indicated interaction at a specific site on the enzyme surface and gave a dissociation constant similar to that from steady-state kinetics, suggesting that the same processes were being observed by both methods and that the active site may be involved. Protection against inactivation was afforded by chloral and NAD together. Stoichiometry of inactivation showed the first 2 equiv per tetramer to abolish the majority of catalytic activity; 4 equiv inactivated both isozymes with complete loss of esterase, NAD-stimulated esterase, and dehydrogenase activities. Peptide mapping of enzyme modified with [carbonyl-14C]bromoacetophenone of CNBr digests (E1) and tryptic digests (E1 and E2) showed one peptide to be preferentially labeled. The above results together with the similarity of bromoacetophenone to the substrate benzaldehyde suggest bromoacetophenone may react with a residue in the active site of aldehyde dehydrogenase. Amino acid analysis of the labeled E1 tryptic fragment indicated reaction with a different peptide from that with which iodoacetamide reacts.
人肝脏醛脱氢酶同工酶E1和E2（EC 1.2.1.3）均可被溴苯乙酮（2-溴-1-苯乙酮）完全且不可逆地灭活。对苯乙酮和氯苯乙酮的稳态动力学研究表明，它们与醛底物作用于同一种酶形式。氯苯乙酮和溴苯乙酮的饱和动力学研究表明，它们在酶表面的特定位点相互作用，其解离常数与稳态动力学研究所得结果相似，这表明两种方法观察到的是相同的过程，且活性位点可能参与其中。水合氯醛和NAD共同提供了对灭活的保护作用。灭活的化学计量学表明，每个四聚体的前2当量可消除大部分催化活性；4当量可使两种同工酶均失活，酯酶、NAD刺激的酯酶和脱氢酶活性完全丧失。用[羰基-¹⁴C]溴苯乙酮修饰的酶（E1经CNBr消化，E1和E2经胰蛋白酶消化）进行肽图谱分析，结果显示有一个肽段被优先标记。上述结果以及溴苯乙酮与底物苯甲醛的相似性表明，溴苯乙酮可能与醛脱氢酶活性位点中的一个残基发生反应。对标记的E1胰蛋白酶片段进行氨基酸分析，结果表明其与碘乙酰胺反应的肽段不同。
