Abstract
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 Val(97) 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.
Highlights
Histidine triad nucleotide-binding proteins (Hint)2 are members of the histidine triad (HIT) protein superfamily of nucleotidyltransferases and hydrolyases [1]
The precise mechanism of action by which it affects tumor development is not well understood, site-directed mutagenesis studies have indicated that the tumor suppressor function of fragile histidine triad (Fhit) is likely not dependent on its Ap3A hydrolase activity [20]
Design of Monomeric Human Hint1—The homodimeric structure of hHint1 is shown in Fig. 1A with Val97 highlighted at the dimer interface
Summary
Histidine triad nucleotide-binding protein; hHint, human histidine triad nucleotide-binding protein 1; AIPA, adenosine 5Ј-indole-3-propionic adenylate; AMPPCP, adenosine 5Ј-(␣,-methylenediphosphate); HIT, histidine triad; MTX, methotrexate; r.m.s. deviation, root mean square deviation; SEC, size exclusion chromatography; TpAd, tryptamine adenosine phosphoramidate monoester; Ap3A, diadenosine P1,P3-triphosphate; Fhit, fragile histidine triad; GalT, galactose-1-phosphate uridyltransferase; DHFR, dihydrofolate reductase. The precise mechanism of action by which it affects tumor development is not well understood, site-directed mutagenesis studies have indicated that the tumor suppressor function of Fhit is likely not dependent on its Ap3A hydrolase activity [20]. GalT is a homodimer with little overall sequence homology with hHint or Fhit proteins, it does share some three-dimensional structure similarity [22]. The mechanism of the phosphoramidase and aminoacylAMP hydrolase activity catalyzed by Hints has been proposed to follow a reaction pathway similar to that for GalT and Fhit [1, 6]. Similar to other homodimeric enzymes, such as bacterial alkaline phosphatase, each monomer contains a well separated active site that does not participate in the dimer interface [25]. The effect of homodimerization on the hydrolysis of lysyl-AMP produced by LysRS was determined
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