Proton transfer dynamics of GART: The pH-dependent catalytic mechanism examined by electrostatic calculations

The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes the t ransfer of a formyl group from formyl tetrahydrofolate (fTHF) to glycinamid e ribonucleotide (GAR), a process that is pH-dependent with pK(a) of simila r to8. Experimental studies of pH-rate profiles of wild-type and site-direc ted mutants of GART have led to the proposal that His108, Asp144, and GAR a re involved in catalysis, with His108 being an acid catalyst, while forming a salt bridge with Asp144, and GAR being a nucleophile to attack the formy l group of fTHF. This model implied a protonated histidine with pK(a) of 9. 7 and a neutral GAR with pK(a) of 6.8. These proposed unusual pK(a)s have l ed us to investigate the electrostatic environment of the active site of GA RT. We have used Poisson-Boltzmann-based electrostatic methods to calculate the pK(a)S of all ionizable groups, using the crystallographic structure o f a ternary complex of GART involving the pseudosubstrate 5-deaza-5,6,7,8-T HF (5dTHF) and substrate GAR. Theoretical mutation and deletion analogs hav e been constructed to elucidate pairwise electrostatic interactions between key ionizable sites within the catalytic site. Also, a construct of a more realistic catalytic site including a reconstructed pseudocofactor with an attached formyl group, in an environment with optimal local van der Waals i nteractions (locally minimized) that imitates closely the catalytic reactan ts, has been used for pK(a) calculations. Strong electrostatic coupling amo ng catalytic residues His108, Asp144, and substrate GAR was observed, which is extremely sensitive to the initial protonation and imidazole ring flip state of His108 and small structural changes. We show that a proton can be exchanged between GAR and His108, depending on their relative geometry and their distance to Asp144, and when the proton is attached on His108, cataly sis could be possible. Using the formylated locally minimized construct of GART, a high pK(a) for His108 was calculated, indicating a protonated histi dine, and a low pK(a) for GAR(NH2) was calculated, indicating that GAR is i n neutral form. Our results are in qualitative agreement with the current m echanistic picture of the catalytic process of GART deduced from the experi mental data, but they do not reproduce the absolute magnitude of the pK(a)s extracted from fits of k(cat)-pH profiles, possibly because the static tim e-averaged crystallographic structure does not describe adequately the dyna mic nature of the catalytic site during binding and catalysis. In addition, a strong effect on the pK(a) of GAR(NH2) is produced by the theoretical mu tations of His108Ala and Asp144Ala, which is not in agreement with the obse rved insensitivity of the pK(a) of GAR(NH2) modeled from the experimental d ata using similar mutations. Finally, we show that important three-way elec trostatic interactions between highly conserved His137, with His108 and Asp 144, are responsible for stabilizing the electrostatic microenvironment of the catalytic site. In conclusion, our data suggest that further detailed c omputational and experimental work is necessary.