A cysteine in the C-terminal region of alanyl-tRNA synthetase is important for aminoacylation activity.
Alanyl-tRNA synthetase (AlaRS) from Escherichia coli is a multimeric enzyme that catalyzes the esterification of alanine to tRNA(Ala) in the ATP-dependent aminoacylation reaction. The functional binding of all three substrates follows Michaelis-Menten kinetics. The role of cysteines in this enzyme has been evaluated via modification of these residues with p-(hydroxymercuri)phenylsulfonic acid, monobromobimane, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The former two reagents induce nearly complete inactivation of AlaRS aminoacylation activity and the release of all tightly bound zinc. In the case of mild DTNB treatment, only two of the six cysteines in AlaRS are modified, with release of all zinc and partial loss of aminoacylation activity. These experiments indicate the importance of one or more cysteines, other than those thought to be coordinated with zinc, in the aminoacylation reaction. Substitution of each of the cysteine residues outside the zinc-binding motif with serine does not disrupt zinc binding. However, the cysteine most removed in primary sequence from the active site (Cys665) is identified as important in the aminoacylation step. Mutation of Cys665 to serine induces a 120-fold decrease in the catalytic efficiency of this enzyme, primarily through a kcat effect, and introduces sigmoidal kinetics (nH = 1.8) with respect to the RNA substrate. The results demonstrate that a simple manipulation in the C-terminal region can introduce positive cooperativity in this otherwise noncooperative enzyme.
Wu
M X
MX
Department of Biochemistry, Loma Linda University School of Medicine, California 92350.
Filley
S J
SJ
Xiong
J
J
Lee
J J
JJ
Hill
K A
KA
D000215:Acylation; D000412:Alanine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D003545:Cysteine / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology*; D008970:Molecular Weight; D016296:Mutagenesis; D015032:Zinc / Q000032:analysis
D000412:Alanine-tRNA Ligase; D015032:Zinc; D003545:Cysteine