Human Hypoxanthine-Guanine Phosphoribosyl Transferase: Molecular Basis of the Enzyme Deficiency States.

Abstract

A complete deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) in man leads to the development of the Lesch-Nyhan syndrome whereas a partial deficiency of the same enzyme leads to a severe form of gout. In an attempt to define the molecular basis of these enzyme-deficiency states, I have investigated the structural and functional properties of HPRT from normal and HPRT-deficient patients. The complete primary structure of human HPRT was defined by sequence analysis of peptide fragments formed by cleavage at arginine, glutamic acid, and methionine residues. The enzyme is 217 residues long with a molecular weight equal to 24,470. The erythrocyte enzyme undergoes two posttranslational modifications in vivo: (1) removal of the NH(,2) terminal methionine followed by acetylation of the free NH(,2)-terminus, and (2) partial deamidation of asparagine 106. A comparison of the amino acid sequence of human HPRT with that of ATP phosphoribosyltransferase from S. typhimurium and glutamine P-Rib-PP amidotransferase from E. coli suggested a functional homology in a sequence of 120 amino acids long (residues 1 to 120 in HPRT). Secondary structure prediction analyses of these shared regions indicated a dinucleotide fold with its characteristic (beta)(alpha)(beta) secondary structural pattern. Five unique structural variants of human HPRT were purified and characterized from unrelated HPRT-deficient patients; HPRT(,Ann Arbor), HPRT(,London), HPRT(,Munich), and HPRT(,Toronto) were isolated from male patients who presented with a severe form of gout, while HPRT(,Kinston) was isolated from a patient with the Lesch-Nyhan syndrome. A sensitive HPLC method was developed to map the tryptic peptides of human HPRT. Four of the enzyme variants were subjected to this analysis and shown to differ from the normal enzyme by a unique tryptic peptide. Sequence analysis of the aberrant peptides revealed single amino acid substitutions in each variant: HPRT(,Toronto), Arg(,50)(--->)Gly; HPRT(,Munich), Ser(,103)(--->)Arg; HPRT(,London), Ser(,109)(--->)Leu; and HPRT(,Kinston), Asp(,193)(--->)Asn. All four amino acid substitutions can be explained by single nucleotide changes in the DNA. The relative location of the first three mutations within the putative dinucleotide fold of human HPRT predicts the associated abnormalities in enzyme function. These patients represent the first in whom specific mutations have been defined at this genetic locus.