Berleth E S, Li J, Braunscheidel J A, Pickart C M
Department of Biochemistry, State University of New York, Buffalo 14214.
Arch Biochem Biophys. 1992 Nov 1;298(2):498-504. doi: 10.1016/0003-9861(92)90441-x.
Aminoacyl-tRNA protein transferases post-translationally aminoacylate protein N-termini. At least in part, these enzymes function to allow a subset of cellular proteins to be targeted for protein degradation. A eukaryotic enzyme of this class, Arg aminoacyl-tRNA protein transferase, arginylates N-terminal Glu or Asp residues of proteins, allowing such proteins to be recognized by a specific ubiquitin-protein ligase. We showed previously that inorganic arsenite, a reagent expected to bind specifically to protein vicinal thiol groups, inhibited Arg aminoacyl-tRNA transferase activity in rabbit reticulocyte lysate (N. S. Klemperer and C. M. Pickart, 1989, J. Biol. Chem. 264, 19245-19252). We now report that a bifunctional arsenoxide reagent, p-[(bromoacetyl)-amino]phenylarsenoxide, is a potent and irreversible inactivator of the same enzyme (K0.5 = 11.5 microM). Bromoacetyl aniline, which lacks the arsenoxide moiety, has no effect. These results show that the transferase has a reactive nucleophile proximal to the site which binds arsenoxides. The related monofunctional arsenoxide reagent, p-aminophenylarsenoxide, is a reversible inhibitor whose potency (K0.5 = 7.7 microM) is 20-fold greater than that of inorganic arsenite. As expected for a mechanism in which p-aminophenylarsenoxide binds to vicinal thiol groups: (i) pretreatment of reticulocyte lysate with a thiol-blocking reagent prevents binding of the transferase to a phenylarsenoxide-Sepharose column; and (ii) inhibition by p-aminophenylarsenoxide is reversed by a competing chemical dithiol, but not by a monothiol reagent. Like the rabbit enzyme, Arg aminoacyl-tRNA protein transferase from the yeast Saccharomyces cerevisiae (expressed in Escherichia coli) is reversibly inhibited by the monofunctional phenylarsenoxide and irreversibly inactivated by the bifunctional phenylarsenoxide (but not by bromoacetylaniline). Thus, a reactive nucleophile proximal to vicinal thiol groups is a conserved feature of the activity of the transferase. We speculate that these groups are catalytic elements in the transferase active site.
氨酰基 - tRNA蛋白转移酶在翻译后对蛋白质N端进行氨酰化修饰。这些酶至少部分地发挥作用，使一部分细胞蛋白能够被靶向进行蛋白质降解。这类真核酶中的一种，精氨酸氨酰基 - tRNA蛋白转移酶，会对蛋白质的N端谷氨酸或天冬氨酸残基进行精氨酸化修饰，从而使这些蛋白质能够被一种特定的泛素 - 蛋白连接酶识别。我们之前曾表明，无机亚砷酸盐是一种预期会特异性结合蛋白质邻位巯基的试剂，它能抑制兔网织红细胞裂解物中的精氨酸氨酰基 - tRNA转移酶活性（N. S. 克莱姆珀勒和C. M. 皮卡尔特，1989年，《生物化学杂志》264卷，19245 - 19252页）。我们现在报告，一种双功能亚砷氧化物试剂，对 - [(溴乙酰基) - 氨基]苯亚砷氧化物，是该同一种酶的一种强效且不可逆的失活剂（K0.5 = 11.5微摩尔）。缺乏亚砷氧化物部分的溴乙酰苯胺则没有作用。这些结果表明，转移酶在与亚砷氧化物结合位点附近有一个反应性亲核试剂。相关的单功能亚砷氧化物试剂，对 - 氨基苯亚砷氧化物，是一种可逆抑制剂，其效力（K0.5 = 7.7微摩尔）比无机亚砷酸盐高20倍。正如对 - 氨基苯亚砷氧化物与邻位巯基结合的机制所预期的那样：（i）用一种巯基阻断试剂预处理网织红细胞裂解物可阻止转移酶与苯亚砷氧化物 - 琼脂糖柱结合；（ii）对 - 氨基苯亚砷氧化物的抑制作用可被一种竞争性化学二硫醇逆转，但不能被单硫醇试剂逆转。与兔酶一样，来自酿酒酵母（在大肠杆菌中表达）的精氨酸氨酰基 - tRNA蛋白转移酶可被单功能苯亚砷氧化物可逆抑制，并被双功能苯亚砷氧化物不可逆失活（但不被溴乙酰苯胺失活）。因此，邻位巯基附近的反应性亲核试剂是转移酶活性的一个保守特征。我们推测这些基团是转移酶活性位点中的催化元件。
