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 phosphoribosyltransferase (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 (k(cat)/K-m) with hypoxanthine as substrate, but with guanine, the mutant enzyme exhibited a more than 50-fold higher k(cat)/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 k(cat)/K-m values for hypoxanthine and guanine compared to wild-type, and replacement of Leu-192 with lie had no significant change in kinetics for either hypoxanthine or PRPP. However, this Ile substitution resulted in an over 15-fold decrease in the k(cat)/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.