Unusual substrate specificity of a chimeric hypoxanthine-guanine phosphoribosyltransferase containing segments from the Plasmodium falciparum and human enzymes

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the phosph oribosylation of hypoxanthine and guanine by transferring the phosphoribosy l moiety from phosphoribosylpyrophosphate (PRPP) on to N9 in the purine bas e, resulting in the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP). Xanthine is an additional substrate for the Plasmodium falciparum HGXPRT. Our aim has been to elucidate structural features in HG PRT that govern substrate specificity. We have addressed this problem by en gineering chimeric HGPRTs, which contain segments from both the parasite an d human enzymes. Four chimeric enzymes were engineered (DS1-DS4), of which the chimeric enzyme, DS1, in which the first 49 residues of human HGPRT wer e replaced with the corresponding residues from the P, falciparum enzyme, e xhibited additional specificity for xanthine, None of the switched residues forms a part of the purine or PRPP binding region in the available crystal structures of HG(X)PRTs, Our data on the chimeric enzyme DS1 provide the f irst evidence that the N-terminal similar to 50 amino acids, although not p roximal to the active site in the crystal structure, can in fact modulate s ubstrate specificity. DS1 exhibits a reduced k(cat) for hypoxanthine and gu anine, while its K-m for these oxopurine bases remains largely unchanged. I ts specific activity for xanthine is comparable with hypoxanthine but five times more than that for guanine, (C) 2000 Academic Press.