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.
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.
D000659:AMP Deaminase / Q000037:antagonists & inhibitors*; D000217:Acyltransferases / Q000037:antagonists & inhibitors*; D058892:Adenosine Deaminase Inhibitors; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D005492:Folic Acid / Q000031:analogs & derivatives*; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000494:pharmacology; D009700:Nucleoside Deaminases / Q000037:antagonists & inhibitors*; D009709:Nucleotide Deaminases / Q000037:antagonists & inhibitors*; D010455:Peptides / Q000494:pharmacology*; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D011099:Polyglutamic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D011624:Pteroylpolyglutamic Acids / Q000494:pharmacology*; D012265:Ribonucleotides / Q000494:pharmacology; D013053:Spectrophotometry
D058892:Adenosine Deaminase Inhibitors; D010455:Peptides; D011624:Pteroylpolyglutamic Acids; D012265:Ribonucleotides; D011099:Polyglutamic Acid; D000620:Aminoimidazole Carboxamide; C014085:methotrexate polyglutamate; D005492:Folic Acid; D019877:Hydroxymethyl and Formyl Transferases; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D000217:Acyltransferases; D009700:Nucleoside Deaminases; D009709:Nucleotide Deaminases; D000659:AMP Deaminase; C031143:AICA ribonucleotide; D008727:Methotrexate