Human AICAR transformylase: Role of the 4-carboxamide of AICAR in binding and catalysis

We have prepared 4-substituted analogues of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to investigate the specificity and mechanism of AICA R transformylase (AICAR Tfase). Of the nine analogues of AICAR studied, onl y one analogue, 5-aminoimidazole-4-thiocarboxamide ribonucleotide, was a su bstrate, and it was converted to 6-mercaptopurine ribonucleotide. The other analogues either did not bind or were competitive inhibitors, the most pot ent being 5-amino-4-nitroimidazole ribonucleotide with a K-i of 0.7 +/- 0.5 mu M. The results show that the 4-carboxamide of AICAR is essential for ca talysis, and it is proposed to assist in mediating proton transfer, catalyz ing the reaction by trapping of the addition compound. AICAR analogues wher e the nitrogen of the 4-carboxamide was derivatized with a methyl or an all ylic group did not bind AICAR Tfase, as determined by pre-steady-state burs t kinetics; however, these compounds were potent inhibitors of IMP cyclohyd rolase (IMP CHase), a second activity of the bifunctional mammalian enzyme (K-i = 0.05 +/- 0.02 mu M for 4-N-allyl-AICAR). It is proposed that the con formation of the carboxamide moiety required for binding to AICAR Tfase is different than the conformation required for binding to IMP CHase, which is supported by inhibition studies of purine ribonucleotides. It is shown tha t 5-formyl-AICAR (FAICAR) is a product inhibitor of AICAR Tfase with K-i of 0.4 +/- 0.1 mu M. We have determined the equilibrium constant of the trans formylase reaction to be 0.024 +/- 0.001, showing that the reaction strongl y favors AICAR and the 10-formyl-folate cofactor. The coupling of the AICAR Tfase and IMP CHase activities on a single polypeptide allows the overall conversion of AICAR to IMP to be favorable by coupling the unfavorable form ation of FAICAR with the highly favorable cyclization reaction. The current kinetic studies have also indicated that the release of FAICAR is the rate -limiting step, under steady-state conditions, in the bifunctional enzyme a nd channeling is not observed between AICAR Tfase and IMP CHase.