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 appa rent homogeneity from human CCRF-CEM leukemia cells, completed with ch romatography on Affi-Gel Blue followed by AICAR-Sepharose 4B. Using a sensitive radioassay, IMP cyclohydrolase has a K-s value for 5-formami doimidazole-4-carboxamide ribotide (FAICAR) at pH 7.4 of 0.87 +/- 0.11 mu M. The following purine nucleotide derivatives were potent competi tive inhibitors of IMP cyclohydrolase: 2-mercaptoinosine 5'-monophosph ate (K-i = 0.094 +/- 0.024 mu M), xanthosine 5'-monophosphate (K-i = 0 .12 +/- 0.01 mu M), 2-fluoroadenine arabinoside 5'-monophosphate (K-i = 0.16 +/- 0.02 mu M), 6-mercaptopurine riboside 5'-monophosphate (K-i = 0.20 +/- 0.02 mu M), adenosine N-1-oxide 5'-monophosphate (K-i = 0. 28 +/- 0.03 mu M), and N-6-(carboxymethyl)adenosine 5'-monophosphate ( K-i = 1.7 +/- 0.42 mu M). The pH dependencies of V-max and V-max/K-s I MP cyclohydrolase are consistent with a single ionizable amino acid re sidue (pK(a) = 7.57 +/- 0.09) of the enzyme which must be unprotonated for catalysis to occur and a residue (pK(a) = 7.57 +/- 0.14) which mu st be unprotonated for FAICAR to bind. The pK(a) 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 obs erved. Chemical modification of IMP cyclohydrolase provides evidence f or arginine and cysteine residues at the active site, and roles for th ese residues in the mechanism of catalysis are proposed.