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Detection of inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase by thioinosinic acid and azathioprine by a new colorimetric assay.

ABSTRACT: The colorimetric assay for 5-aminoimidazole-4-carboxamide ribotide (AICAR) transformylase (phosphoribosylamino-imidazolecarboxamide formyltransferase; EC 2.1.2.3) has been extensively modified. The modified assay is based upon the short-term permanganate oxidation of the folate product, tetrahydrofolate (H4folate) to p-aminobenzoyl glutamate (pABG). The modified assay was used to detect the transformylase activity in crude extracts of peripheral-blood mononuclear cells (PBMCs). Azathioprine and its metabolite, thioinosinic acid (tIMP), are competitive inhibitors (with respect to AICAR) of the chicken liver transformylase and the transformylase from PBMCs of the MRL/lpr mouse, an animal model of systemic autoimmune disease. The Ki values of tIMP and azathioprine for the chicken liver enzyme are 39 +/- 4 microM and 120 +/- 10 microM, whereas the Ki values for the enzyme from PBMCs of the MRL/lpr mouse are 110 +/- 20 microM and 90 +/- 14 microM respectively. The anti-inflammatory drugs ibuprofen and naproxen are also inhibitors of the transformylase.

SUBMITTER: Ha T

PROVIDER: S-EPMC1149705 | biostudies-other | 1990 Dec

REPOSITORIES: biostudies-other

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Project description:With the use of a continuous spectrophotometric assay and initial rates determined by the method of Waley [Biochem. J. (1981) 193, 1009-1012] methotrexate was found to be a non-competitive inhibitor, with Ki(intercept) = 72 microM and Ki(slope) = 41 microM, of 5-aminoimidazole-4-carboxamide ribotide transformylase, whereas a polyglutamate of methotrexate containing three gamma-linked glutamate residues was a competitive inhibitor, with Ki = 3.15 microM. Pentaglutamates of folic acid and 10-formylfolic acid were also competitive inhibitors of the transformylase, with Ki values of 0.088 and 1.37 microM respectively. Unexpectedly, the pentaglutamate of 10-formyldihydrofolic acid was a good substrate for the transformylase, with a Km of 0.51 microM and a relative Vmax. of 0.72, which compared favourably with a Km of 0.23 microM and relative Vmax. of 1.0 for the tetrahydro analogue. An analysis of the progress curve of the transformylase-catalysed reaction with the above dihydro coenzyme revealed that the pentaglutamate of dihydrofolic acid was a competitive product inhibitor, with Ki = 0.14 microM. The continuous spectrophotometric assay for adenosine deaminase based on change in the absorbance at 265 nm was shown to be valid with adenosine concentrations above 100 microM, which contradicts a previous report [Murphy, Baker, Behling & Turner (1982) Anal. Biochem. 122, 328-337] that this assay was invalid above this concentration. With the spectrophotometric assay, 5-aminoimidazole-4-carboxamide riboside was found to be a competitive inhibitor of adenosine deaminase, with (Ki = 362 microM), whereas the ribotide was a competitive inhibitor of 5'-adenylate deaminase, with Ki = 1.01 mM. Methotrexate treatment of susceptible cells results in (1) its conversion into polyglutamates, (2) the accumulation of oxidized folate polyglutamates, and (3) the accumulation of 5-aminoimidazole-4-carboxamide riboside and ribotide. The above metabolic events may be integral elements producing the cytotoxic effect of this drug by (1) producing tighter binding of methotrexate to folate-dependent enzymes, (2) producing inhibitors of folate-dependent enzymes from their tetrahydrofolate coenzymes, and (3) trapping toxic amounts of adenine nucleosides and nucleotides as a result of inhibition of adenosine deaminase and 5'-adenylate deaminase respectively.

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Detection of inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase by thioinosinic acid and azathioprine by a new colorimetric assay.

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