A single amino acid substitution in the human and a bacterial hypoxanthine phosphoribosyltransferase modulates specificity for the binding of guanine.
Early studies involving purine salvage in Salmonella typhimurium resulted in the isolation and identification of a mutant strain possessing a genetically modified hypoxanthine phosphoribosyl-transferase (HPRT) with enhanced substrate specificity for guanine [Benson, C. E., and Gots, J. S. (1975) J. Bacteriol. 121, 77-82]. To explore the molecular basis for this altered substrate specificity in the mutant hpt gene product, degenerate oligonucleotide primers, designed according to the N- and C-termini of the HPRT of Escherichia coli, were used in polymerase chain reactions to amplify both the mutant and wild-type S. typhimurium hpt genes from genomic DNA. Analysis of the deduced amino acid sequences revealed that a single base mutation resulted in the encoding of a Thr in the mutant HPRT, instead of an Ile found in the wild-type enzyme, at a position analogous to position 192 (Leu-192) of the human HPRT. Comparison of kinetic data for purified recombinant mutant and wild-type HPRTs showed no difference in the overall catalytic efficiency (kcat/K(m)) with hypoxanthine as substrate, but with guanine, the mutant enzyme exhibited a more than 50-fold higher kcat/K(m) largely as a result of a decrease of nearly 2 orders of magnitude in K(m). Involvement in substrate binding of the cognate amino acid at position 192 in the human HPRT was investigated using site-directed mutagenesis. Mutation of Leu-192 to Thr did not significantly alter kcat/K(m) values for hypoxanthine and guanine compared to wild-type, and replacement of Leu-192 with Ile had no significant change in kinetics for either hypoxanthine or PRPP. However, this Ile substitution resulted in an over 15-fold decrease in the kcat/K(m) for guanine due to a greater than 15-fold increase in K(m). These results demonstrate that a single active site amino acid substitution in HPRTs can significantly alter the specificity for binding guanine.
Lee
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CC
Laboratory of Molecular Parasitology & Drug Design, University of North Carolina at Chapel Hill 27599-7360, USA.
Craig
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SP
Eakin
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AE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D017931:DNA Primers; D011994:Recombinant Proteins; D007041:Hypoxanthine Phosphoribosyltransferase