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-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.

Pathobiology of NPM-ALK and variant fusion genes in anaplastic large cell lymphoma and other lymphomas

“…22,23 The fusion gene is detectable by RT-PCR (with identical fusion transcripts), the reciprocal (ALK-ATIC) fusion cDNA was not found. The amino terminus of ATIC, a bifunctional homodimeric enzyme that catalyzes the penultimate and final steps of de novo purine nucleotide biosynthesis, [193][194][195] fuses with the intracellular portion of the ALK receptor tyrosine kinase. The chimeric protein is localized in the cytoplasm and is constitutively tyrosine phosphorylated.…”

Human 5-Aminoimidazole-4-carboxamide Ribonucleotide Transformylase/Inosine 5′-Monophosphate Cyclohydrolase

“…Each assay was initiated by rapid mixing of the substrates with the assay solution in the cuvette. The 10-f-FH 4 was synthesized as described previously (8,15,16) from (6S)5-formyl-5,6,7,8-tetrahydrofolic acid (leucovorin) purchased from Schircks Laboratories. AICAR was purchased from Sigma.…”

“…Thus it is important to determine how the substrates influence the oligomeric state and which oligomeric form of ATIC is active for both the AICAR TFase and IMPCHase activities. To determine whether the monomer/dimer equilibrium of ATIC is shifted in the presence of one or more of its substrates, the sedimentation equilibrium experiments were repeated with solutions containing AICAR, 10-f-FH 4 , or AICAR and the folate analog DDATHF at concentrations 10 times the K m or the K i in the case of DDATHF (4,7,8,11,21). A global nonlinear analysis was carried out as above using two initial concentrations at three rotor speeds for each of the above conditions using Winnl106.…”

“…The human form of ATIC (hATIC) was first cloned in our laboratory from a hepatoma cDNA library (8). Sugita et al (9) later found an additional form of hATIC cloned from a human placenta cDNA library.…”

“…Enzyme cross-linking experiments using both yeast isoforms and the avian form of the enzyme suggest that ATIC may form a dimer (10,11). Gel filtration experiments using recombinant human enzyme conducted by Rayl et al (8) suggested that ATIC exists as a monomer under the conditions tested (8). These apparently conflicting conclusions may actually both be valid, because the results depend greatly on the experimental conditions used to study the oligomeric properties of a protein.…”