Kraut D, Goff H, Pai R K, Hosea N A, Silman I, Sussman J L, Taylor P, Voet J G
Department of Chemistry, Swarthmore College, Swarthmore, PA 19081, USA.
Mol Pharmacol. 2000 Jun;57(6):1243-8.
Acetylcholinesterase (AChE), a serine hydrolase, is potentially susceptible to inactivation by phenylmethylsulfonyl fluoride (PMSF) and benzenesulfonyl fluoride (BSF). Although BSF inhibits both mouse and Torpedo californica AChE, PMSF does not react measurably with the T. californica enzyme. To understand the residue changes responsible for the change in reactivity, we studied the inactivation of wild-type T. californica and mouse AChE and mutants of both by BSF and PMSF both in the presence and absence of substrate. The enzymes investigated were wild-type mouse AChE, wild-type T. californica AChE, wild-type mouse butyrylcholinesterase, mouse Y330F, Y330A, F288L, and F290I, and the double mutant T. californica F288L/F290V (all mutants given T. californica numbering). Inactivation rate constants for T. californica AChE confirmed previous reports that this enzyme is not inactivated by PMSF. Wild-type mouse AChE and mouse mutants Y330F and Y330A all had similar inactivation rate constants with PMSF, implying that the difference between mouse and T. californica AChE at position 330 is not responsible for their differing PMSF sensitivities. In addition, butyrylcholinesterase and mouse AChE mutants F288L and F290I had increased rate constants ( approximately 14 fold) over those of wild-type mouse AChE, indicating that these residues may be responsible for the increased sensitivity to inactivation by PMSF of butyrylcholinesterase. The double mutant T. californica AChE F288L/F290V had a rate constant nearly identical with the rate constant for the F288L and F290I mouse mutant AChEs, representing an increase of approximately 4000-fold over the T. californica wild-type enzyme. It remains unclear why these two positions have more importance for T. californica AChE than for mouse AChE.
乙酰胆碱酯酶（AChE）是一种丝氨酸水解酶，可能易被苯甲基磺酰氟（PMSF）和苯磺酰氟（BSF）灭活。尽管BSF可抑制小鼠和加州电鳐的AChE，但PMSF与加州电鳐的酶没有可测量的反应。为了解导致反应性变化的残基变化，我们研究了野生型加州电鳐和小鼠AChE以及两者的突变体在有底物和无底物存在的情况下被BSF和PMSF灭活的情况。所研究的酶包括野生型小鼠AChE、野生型加州电鳐AChE、野生型小鼠丁酰胆碱酯酶、小鼠Y330F、Y330A、F288L和F290I，以及双突变体加州电鳐F288L/F290V（所有突变体均采用加州电鳐的编号）。加州电鳐AChE的失活速率常数证实了先前的报道，即该酶不会被PMSF灭活。野生型小鼠AChE以及小鼠突变体Y330F和Y330A与PMSF的失活速率常数都相似，这意味着小鼠和加州电鳐AChE在330位的差异并非其对PMSF敏感性不同的原因。此外，丁酰胆碱酯酶和小鼠AChE突变体F288L和F290I的速率常数比野生型小鼠AChE增加了（约14倍），表明这些残基可能是丁酰胆碱酯酶对PMSF灭活敏感性增加的原因。双突变体加州电鳐AChE F288L/F290V的速率常数与F288L和F290I小鼠突变体AChE的速率常数几乎相同，比加州电鳐野生型酶增加了约4000倍。目前尚不清楚为什么这两个位置对加州电鳐AChE比小鼠AChE更重要。
