5-Aminoimidazole-4-carboxamide ribotide transformylase-IMP cyclohydrolase from human CCRF-CEM leukemia cells: purification, pH dependence, and inhibitors.
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.
Szabados
E
E
Department of Biochemistry, University of Sydney, NSW, Australia.
Hindmarsh
E J
EJ
Phillips
L
L
Duggleby
R G
RG
Christopherson
R I
RI
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D007938:Leukemia / Q000201:enzymology*; D009709:Nucleotide Deaminases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D010766:Phosphorylation; D011687:Purines / Q000494:pharmacology; D013053:Spectrophotometry; D014407:Tumor Cells, Cultured
D009711:Nucleotides; D011687:Purines; D001120:Arginine; D019877:Hydroxymethyl and Formyl Transferases; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000217:Acyltransferases; D009709:Nucleotide Deaminases; C021436:IMP cyclohydrolase; D003545:Cysteine