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J. Biol. Chem., Vol. 282, Issue 7, 4719-4727, February 16, 2007

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Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins^*

Tsui-Fen Chou and Carston R. Wagner¹

From the Departments of Medicinal Chemistry and Chemistry, University of Minnesota, Minneapolis, Minnesota 55455

Histidine triad nucleotide binding proteins (Hints) are the most ancient members of the histidine triad protein superfamily of nucleotidyltransferases and hydrolyases. Protein-protein interaction studies have found that complexes of the transcription factors MITF or USF2 and lysyl-tRNA synthetase (LysRS) are associated with human Hint1. Therefore, we hypothesized that lysyl-AMP or the LysRS·lysyl-AMP may be a native substrate for Hints. To explore the biochemical relationship between Hint1 and LysRS, a series of catalytic radiolabeling, mutagenesis, and kinetic experiments was conducted with purified LysRSs and Hints from human and Escherichia coli. After incubation of the E. coli or human LysRS with Hints and [-³²P]ATP, but not [-³²P]GTP, ³²P-labeled Hints were observed. By varying time and the concentrations of lysine, Mg²⁺, or LysRS, the adenylation of Hint was found to be dependent on the formation of lysyl-AMP. Site-directed mutagenesis studies of the active site histidine triad revealed that Hint labeling could be abolished by substitution of either His-101 of E. coli hinT or His-112 of human Hint1 by either alanine or glycine. Ap₄A, believed to be synthesized by LysRS in vivo, and Zn²⁺ were shown to inhibit the formation of Hint-AMP with an IC₅₀ value in the low micromolar range. Consistent with pyrophosphate being an inhibitor for aminoacyl-tRNA synthetase, incubations in the presence of pyrophosphatase resulted in enhanced formation of Hint-AMP. These results demonstrate that the lysyl-AMP intermediate formed by LysRS is a natural substrate for Hints and suggests a potential highly conserved regulatory role for Hints on LysRS and possibly other aminoacyl-tRNA synthetases.

Received for publication, November 13, 2006 , and in revised form, December 8, 2006.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ To whom correspondence should be addressed: University of Minnesota, Dept. of Medicinal Chemistry, 8-174 Weaver Densford Hall, 308 Harvard St. S.E., Minneapolis, MN 55455. Tel.: 612-625-2614; Fax: 612-624-0139; E-mail: wagne003{at}umn.edu.

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				T.-F. Chou, I. B. Tikh, B. A. C. Horta, B. Ghosh, R. B. De Alencastro, and C. R. Wagner Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate J. Biol. Chem., May 18, 2007; 282(20): 15137 - 15147. [Abstract] [Full Text] [PDF]