The Human purH Gene Product, 5-Aminoimidazole-4-carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase

Abstract

We report here the cloning and sequencing of the cDNA, purification, steady state kinetic analysis, and truncation mapping studies of the human 5-aminoimi-dazole-4-carboxamide ribonucleotide formyltransfer-ase/IMP cyclohydrolase (AICARFT/IMPCHase). These enzyme activities catalyze the penultimate and the final steps of de novo purine biosynthesis, respectively. 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 length encoding for a 591-amino acid polypeptide (M r ‫؍‬ 64,425). The human 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-formyltetrahy-drofolate are 16.8 ␮M ؎ 1.5 and 60.2 ␮M ؎ 5.0, respec-tively, for the cloned, purified human enzyme. A 10-amino acid sequence within the COOH-terminal portion of human AICARFT/IMPCHase has some degree of ho-mology to a previously noted " folate binding site. " Site-directed mutagenesis studies indicate that this se-quence plays no role in enzymatic activity. We have constructed truncation mutants which demonstrate that each of the two enzyme activities can be expressed independent of the other. IMPCHase and AICARFT ac-tivities are located within the NH 2 -terminal 223 and COOH-terminal 406 amino acids, respectively. The trun-cation mutant possessing AICARFT activity displays steady state kinetic parameters identical to those of the holoenzyme. Aminoimidazole ribonucleotide formyltransferase (AICARFT)