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 transformylase (AICAR Tfase) is evaluated with pH dependent kinetics, site-directed mutagenesis, and quantum chemical calculations. The chemistry step, represented by the burst rates, was not pH-dependent, which is consistent with our proposed mechanism that the 4-carboxamide of AICAR assists proton shuttling. Quantum chemical calculations on a model system of 5-amino-4-carboxamide imidazole (AICA) and formamide using the B3LYP/6-31G level of theory confirmed that the 4-carboxamide participated in the proton-shuttling mechanism. The result also indicated that the amide-assisted mechanism is concerted such that the proton transfers from the 5-amino group to the formamide are simultaneous with nucleophilic attack by the 5-amino group. Because the process does not lead to a kinetically stable intermediate, the intramolecular proton transfer from the 5-amino group through the 4-carboxamide to the formamide proceeds in the same transition state. Interestingly, the calculations predicted that protonation of the N3 of the imidazole of AICA would reduce the energy barrier significantly. However, the pK(a) of the imidazole of AICAR was determined to be 3.23 +/- 0.01 by NMR titration, and AICAR is likely to bind to the enzyme with its imidazole in the free base form. An alternative pathway was suggested by modeling Lys266 to have a hydrogen-bonding interaction with the N3 of the imidazole of AICAR. Lys266 has been implicated in catalysis based on mutagenesis studies and the recent X-ray structure of AICAR Tfase. The quantum chemical calculations on a model system that contains AICA complexed with CH3NH3+ as a mimic of the Lys residue confirmed that such an interaction lowered the activation energy of the reaction and likewise implicated the 4-carboxamide. To experimentally verify this hypothesis, we prepared the K266R mutant and found that its kcat is reduced by 150-fold from that of the wild type without changes in substrate and cofactor Km values. The kcat-pH profile indicated virtually no pH-dependence in the pH range 6-10.5. The results suggest that the ammonium moiety of Lys or Arg is important in catalysis, most likely acting as a general 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 implicated in catalysis. The mutant enzyme showed no detectable activity while retaining its binding affinity for substrate, indicating that it plays a critical 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 imidazole of AICAR.
Shim
J H
JH
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Wall
M
M
Benkovic
S J
SJ
Díaz
N
N
Suárez
D
D
Merz
K M
KM
D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D006863:Hydrogen-Ion Concentration; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D007093:Imidazoles; D019877:Hydroxymethyl and Formyl Transferases; D051027:Phosphoribosylaminoimidazolecarboxamide Formyltransferase