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, steady state kinetic analysis, and truncation mapping studies of the human 5-aminoimidazole- 4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (AICARFT/IMPCHase). These 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 polypeptic (Mr = 64,425). The human and avian purH cDNAs are 75 and 81% similar on the nucleotide and amino acid sequence level, respectively. The Km values for AICAR and (6R,6S)10-formyltetrahydrofolate are 16.8 microM +/- 1.5 and 60.2 microM +/- 5.0, respectively, for the cloned, purified human enzyme. A 10-amino acid sequence within the COOH-terminal portion of human AICARFT/IMPCHase has some degree of homology to a previously noted "folate binding site." Site directed mutagenesis studies indicate that this sequence 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 activities 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 holoenzyme.
Rayl
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EA
Department of Pediatrics, Yale University, New Haven, Connecticut 06510, USA.
Moroson
B A
BA
Beardsley
G P
GP
D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D016384:Consensus Sequence; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D005575:Formyltetrahydrofolates / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009709:Nucleotide Deaminases / Q000235:genetics* / Q000378:metabolism; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D017931:DNA Primers; D018076:DNA, Complementary; D005575:Formyltetrahydrofolates; D012333:RNA, Messenger; D019877:Hydroxymethyl and Formyl Transferases; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000217:Acyltransferases; D009709:Nucleotide Deaminases; C021436:IMP cyclohydrolase