BIOMAT Database Logo BIOMAT Database Logo

5-Aminoimidazole-4-carboxamide ribotide transformylase-IMP cyclohydrolase from human CCRF-CEM leukemia cells: purification, pH dependence, and inhibitors. 1994

E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
Department of Biochemistry, University of Sydney, NSW, Australia.

The bifunctional enzyme 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase-IMP cyclohydrolase has been purified 780-fold to apparent homogeneity from human CCRF-CEM leukemia cells, completed with chromatography on Affi-Gel Blue followed by AICAR-Sepharose 4B. Using a sensitive radioassay, IMP cyclohydrolase has a Ks value for 5-formamidoimidazole-4-carboxamide ribotide (FAICAR) at pH 7.4 of 0.87 +/- 0.11 microM. The following purine nucleotide derivatives were potent competitive inhibitors of IMP cyclohydrolase: 2-mercaptoinosine 5'-monophosphate (Ki = 0.094 +/- 0.024 microM), xanthosine 5'-monophosphate (Ki = 0.12 +/- 0.01 microM), 2-fluoroadenine arabinoside 5'-monophosphate (Ki = 0.16 +/- 0.02 microM), 6-mercaptopurine riboside 5'-monophosphate (Ki = 0.20 +/- 0.02 microM), adenosine N1-oxide 5'-monophosphate (Ki = 0.28 +/- 0.03 microM), and N6-(carboxymethyl)adenosine 5'-monophosphate (Ki = 1.7 +/- 0.42 microM). The pH dependencies of Vmax and Vmax/Ks values for IMP cyclohydrolase are consistent with a single ionizable amino acid residue (pKa = 7.57 +/- 0.09) of the enzyme which must be unprotonated for catalysis to occur and a residue (pKa = 7.57 +/- 0.14) which must be unprotonated for FAICAR to bind. The pKa values of 5.81 +/- 0.03 and 9.41 +/- 0.04 determined for FAICAR indicate that ionization of the substrate does not contribute significantly to the pH effects observed. Chemical modification of IMP cyclohydrolase provides evidence for arginine and cysteine residues at the active site, and roles for these residues in the mechanism of catalysis are proposed.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D007938 Leukemia A progressive, malignant disease of the blood-forming organs, characterized by distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemias were originally termed acute or chronic based on life expectancy but now are classified according to cellular maturity. Acute leukemias consist of predominately immature cells; chronic leukemias are composed of more mature cells. (From The Merck Manual, 2006) Leucocythaemia,Leucocythemia,Leucocythaemias,Leucocythemias,Leukemias
D009709 Nucleotide Deaminases Catalyze the hydrolysis of nucleotides with the elimination of ammonia. Deaminases, Nucleotide
D009711 Nucleotides The monomeric units from which DNA or RNA polymers are constructed. They consist of a purine or pyrimidine base, a pentose sugar, and a phosphate group. (From King & Stansfield, A Dictionary of Genetics, 4th ed) Nucleotide
D010766 Phosphorylation The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety. Phosphorylations
D011687 Purines A series of heterocyclic compounds that are variously substituted in nature and are known also as purine bases. They include ADENINE and GUANINE, constituents of nucleic acids, as well as many alkaloids such as CAFFEINE and THEOPHYLLINE. Uric acid is the metabolic end product of purine metabolism.
D003545 Cysteine A thiol-containing non-essential amino acid that is oxidized to form CYSTINE. Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D006863 Hydrogen-Ion Concentration The normality of a solution with respect to HYDROGEN ions; H+. It is related to acidity measurements in most cases by pH pH,Concentration, Hydrogen-Ion,Concentrations, Hydrogen-Ion,Hydrogen Ion Concentration,Hydrogen-Ion Concentrations

Related Publications

E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
Radioassay of bifunctional 5-aminoimidazole-4-carboxamide ribotide transformylase-IMP cyclohydrolase by thin-layer chromatography.
September 1994, Analytical biochemistry,
E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
Specificity of 4-aminoimidazole-5-carboxamide ribotide transformylase of Escherichia coli.
September 1971, The Biochemical journal,
E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5'-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide.
May 1986, The Biochemical journal,
E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
Human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine 5'-monophosphate cyclohydrolase. A bifunctional protein requiring dimerization for transformylase activity but not for cyclohydrolase activity.
March 2001, The Journal of biological chemistry,
E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
Antifolates induce primary inhibition of the de novo purine pathway prior to 5-aminoimidazole-4-carboxamide ribotide transformylase in leukemia cells.
May 1991, Biochemistry international,
E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
Characterization of two 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase isozymes from Saccharomyces cerevisiae.
July 2000, The Journal of biological chemistry,
E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
Crystal structures of human bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase/IMP cyclohydrolase in complex with potent sulfonyl-containing antifolates.
April 2004, The Journal of biological chemistry,
E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
Characterization of molecularly cloned human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase.
August 1997, Journal of biochemistry,
E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
De novo purine nucleotide biosynthesis: cloning, sequencing and expression of a chicken PurH cDNA encoding 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase-IMP cyclohydrolase.
October 1991, Gene,
E Szabados, and E J Hindmarsh, and L Phillips, and R G Duggleby, and R I Christopherson
The human purH gene product, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase. Cloning, sequencing, expression, purification, kinetic analysis, and domain mapping.
January 1996, The Journal of biological chemistry,
Copied contents to your clipboard!