A REACTIVE NUCLEOPHILE PROXIMAL TO VICINAL THIOLS IS AN EVOLUTIONARILY CONSERVED FEATURE IN THE MECHANISM OF ARG AMINOACYL-TRANSFER RNA PROTEIN TRANSFERASE
A REACTIVE NUCLEOPHILE PROXIMAL TO VICINAL THIOLS IS AN EVOLUTIONARILY CONSERVED FEATURE IN THE MECHANISM OF ARG AMINOACYL-TRANSFER RNA PROTEIN TRANSFERASE
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DOI：
10.1016/0003-9861(92)90441-x
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发表时间：
1992-11-01
影响因子：
3.9
通讯作者：
PICKART, CM
中科院分区：
文献类型：
--
作者：
BERLETH, ES;LI, J;PICKART, CM
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 μM). 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 μM) is 20-fold greater than that of inorganic arsenite. As expected for a mechanism in whichp-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 byp-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 yeastSaccharomyces cerevisiae(expressed inEscherichia 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.
