THE HUMAN PURH GENE-PRODUCT, 5-AMINOIMIDAZOLE-4-CARBOXAMIDE RIBONUCLEOTIDE FORMYLTRANSFERASE IMP CYCLOHYDROLASE - CLONING, SEQUENCING, EXPRESSION, PURIFICATION, KINETIC-ANALYSIS, AND DOMAIN MAPPING

We report here the cloning and sequencing of the cDNA, purification, s teady state kinetic analysis, and truncation mapping studies of the hu man 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IM P cyclohydrolase (AICARFT/IMPCHase). These enzyme activities catalyze the penultimate and the final steps of de novo purine biosynthesis, re spectively. In all species of both prokaryotes and eukaryotes studied, these two activities are present on a single bifunctional polypeptide encoded on the purH gene. The human purH cDNA is 1776 base pairs in l ength encoding for a 591-amino acid polypeptide (M(r) = 64,425). The h uman and avian purH cDNAs are 75 and 81% similar on the nucleotide and amino acid sequence level, respectively. The K-m values for AICAR and (6R,6S) 10-formyltetrahydrofolate are 16.8 mu M +/- 1.5 and 60.2 mu M +/- 5.0, respectively, for the cloned, purified human enzyme. A 10-am ino acid sequence within the COOH-terminal portion of human AICARFT/IM PCHase has some degree of homology to a previously noted ''folate bind ing site.'' Site-directed mutagenesis studies indicate that this seque nce plays no role in enzymatic activity, We have constructed truncatio n mutants which demonstrate that each of the two enzyme activities can be expressed independent of the other. IMPCHase and AICARFT activitie s are located within the NH2-terminal 223 and COOH-terminal 406 amino acids, respectively. The truncation mutant possessing AICARFT activity displays steady state kinetic parameters identical to those of the ho loenzyme.