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J. Biol. Chem., Vol. 282, Issue 20, 15137-15147, May 18, 2007

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Engineered Monomeric Human Histidine Triad Nucleotide-binding Protein 1 Hydrolyzes Fluorogenic Acyl-adenylate and Lysyl-tRNA Synthetase-generated Lysyl-adenylate^*

Tsui-Fen Chou, Ilya B. Tikh, Bruno A. C. Horta, Brahma Ghosh, Ricardo B. De Alencastro, and Carston R. Wagner^¶1

From the Departments of Medicinal Chemistry and ^¶Chemistry, University of Minnesota, Minneapolis, Minnesota 55455 and the Physical Organic Chemistry Group, Departamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, Rio de Janeiro 21941-909, Brazil

Hint1 is a homodimeric protein and member of the ubiquitous HIT superfamily. Hint1 catalyzes the hydrolysis of purine phosphoramidates and lysyl-adenylate generated by lysyl-tRNA synthetase (LysRS). To determine the importance of homodimerization on the biological and catalytic activity of Hint1, the dimer interface of human Hint1 (hHint1) was destabilized by replacement of Val⁹⁷ of hHint1 with Asp, Glu, or Arg. The mutants were shown to exist as monomers in solution by a combination of size exclusion chromatograph, static light scattering, and chemically induced dimerization studies. Circular dichroism studies revealed little difference between the stability of the V97D, V97E, and wild-type hHint1. Relative to wild-type and the V97E mutant, however, significant perturbation of the V97D mutant structure was observed. hHint1 was shown to prefer 3-indolepropionic acyl-adenylate (AIPA) over tryptamine adenosine phosphoramidate monoester (TpAd). Wild-type hHint1 was found to be 277- and 1000-fold more efficient (k_cat/K_m values) than the V97E and V97D mutants, respectively. Adenylation of wild-type, V97D, and V97E hHint1 by human LysRS was shown to correlate with the mutant k_cat/K_m values using 3-indolepropionic acyl-adenylate as a substrate, but not tryptamine adenosine phosphoramidate monoester. Significant perturbations of the active site residues were not detected by molecular dynamics simulations of the hHint1s. Taken together, these results demonstrate that for hHint1; 1) the efficiency (k_cat/K_m) of acylated AMP hydrolysis, but not maximal catalytic turnover (k_cat), is dependent on homodimerization and 2) the hydrolysis of lysyl-AMP generated by LysRS is not dependent on homodimerization if the monomer structure is similar to the wild-type structure.

Received for publication, July 21, 2006 , and in revised form, February 20, 2007.

^* This work was supported in part by a University of Minnesota Faculty Academic Health Center Development Grant and a grant from the National Institutes of Health. 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 Tables 1-2 and Figs. S1-S2.

¹ To whom correspondence should be addressed: 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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