Evaluation of the catalytic mechanism of AICAR transformylase by pH-dependent kinetics, mutagenesis, and quantum chemical calculations

The catalytic mechanism of 5-aminoimidazole-4-carboxamide ribonucleotide tr ansformylase (AICAR Tfase) is evaluated with pH dependent kinetics, site-di rected mutagenesis, and quantum chemical calculations. The chemistry step, represented by the burst rates, was not pH-dependent, which is consistent w ith our proposed mechanism that the 4-carboxamide of AICAR assists proton s huttling. Quantum chemical calculations on a model system of 5-amino-4-carb oxamide imidazole (AICA) acid formamide using the B3LYP/6-31G* level of the ory confirmed that the 4-carboxamide participated in the proton-shuttling m echanism. The result also indicated that the amide-assisted mechanism is co ncerted such that the proton transfers from the 5-amino group to the formam ide are simultaneous with nucleophilic attack by the 5-amino group. Because the process does not lead to a kinetically stable intermediate, the intram olecular proton transfer from the 5-amino group through the 4-carboxamide t o the formamide proceeds in the same transition state. Interestingly, the c alculations predicted that protonation of the N3 of the imidazole of AICA w ould reduce the energy barrier significantly. However, the pK(a) of the imi dazole of AICAR was determined to be 3.23 +/- 0.01 by NMR titration, and AI CAR is likely to bind to the enzyme with its imidazole in the free base for m. An alternative pathway was suggested by modeling Lys266 to have a hydrog en-bonding interaction with the N3 of the imidazole of AICAR, Lys266 has be en implicated in catalysis based on mutagenesis studies and the recent X-ra y structure of AICAR Tfase, The quantum chemical calculations on a model sy stem that contains AICA complexed with CH3NH3+ as a mimic of the Lys residu e confirmed that such an interaction lowered the activation energy of the r eaction and likewise implicated the 4-carboxamide. To experimentally verify this hypothesis, we prepared the K266R mutant and found that its k(cat) is reduced by 150-fold from that of the wild type without changes in substrat e and cofactor K-m values. The k(cat)-pH profile indicated virtually no pH- dependence in the pH range 6-10.5. The results suggest that the ammonium mo iety of Lys or Arg is important in catalysis, most likely acting as a gener al acid catalyst with a pK(a) value greater than 10.5. The H267A mutant was also prepared since His267 has been found in the active site and implicate d in catalysis. The mutant enzyme showed no detectable activity while retai ning its binding affinity for substrate, indicating that it plays a critica l role in catalysis. We propose that His267 interacts with Lys266 to aid in the precise positioning of the general acid catalyst to the N3 of the imid azole of AICAR.