The catalytic mechanism of glyceraldehyde 3-phosphate dehydrogenase from Trypanosoma cruzi elucidated via the QM/MM approach.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been identified as a key enzyme involved in glycolysis processes for energy production in the Trypanosoma cruzi parasite. This enzyme catalyses the oxidative phosphorylation of glyceraldehyde 3-phosphate (G3P) in the presence of inorganic phosphate (Pi) and nicotinamide adenosine dinucleotide (NAD+). The catalytic mechanism used by GAPDH has been intensively investigated. However, the individual roles of Pi and the C3 phosphate of G3P (Ps) sites, as well as some residues such as His194 in the catalytic mechanism, remain unclear. In this study, we have employed Molecular Dynamics (MD) simulations within hybrid quantum mechanical/molecular mechanical (QM/MM) potentials to obtain the Potential of Mean Force of the catalytic oxidative phosphorylation mechanism of the G3P substrate used by GAPDH. According to our results, the first stage of the reaction (oxidoreduction) takes place in the Pi site (energetically more favourable), with the formation of oxyanion thiohemiacetal and thioacylenzyme intermediates without acid-base assistance of His194. Analysis of the interaction energy by residues shows that Arg249 has an important role in the ability of the enzyme to bind the G3P substrate, which interacts with NAD+ and other important residues, such as Cys166, Glu109, Thr167, Ser247 and Thr226, in the GAPDH active site. Finally, the inhibition mechanism of the GAPDH enzyme by the 3-(p-nitrophenoxycarboxyl)-3-ethylene propyl dihydroxyphosphonate inhibitor was investigated in order to contribute to the design of new inhibitors of GAPDH from Trypanosoma cruzi.