A non-active site mutation in human hypoxanthine guanine phosphoribosyltransferase expands substrate specificity.
Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) lacks the ability to phosphoribosylate xanthine, a property exhibited by HGPRTs from many parasitic protozoa. Using random mutagenesis we have obtained a mutant, F36L, of human HGPRT that phosphoribosylates xanthine. Examination of the structure indicates that F36 does not make direct contact with the purine, but long-range modulation via loop IV, a segment contacting purine at C2 position, could influence substrate specificity. Expanded substrate specificity to include xanthine probably arises from increased flexibility of loop IV as a consequence of mutation at F36. Mutation of the corresponding residue, L44 in Plasmodium falciparum HGPRT, also results in alteration of K(m) and k(cat) for xanthine, substantiating its role in affecting purine base affinity. Our studies show that mutation of this residue in the core of the protein also affects the stability of both enzymes.
Raman
Jayalakshmi
J
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, India.
Sumathy
K
K
Anand
Ranjith P
RP
Balaram
Hemalatha
H
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000201:enzymology; D011487:Protein Conformation; D013379:Substrate Specificity
D017931:DNA Primers; D007041:Hypoxanthine Phosphoribosyltransferase