Understanding Enzymatic Hydrolysis Mechanisms using Computer Simulations

• Applicant: Professor Dr. Dominik Marx
• Subject Area: Biophysics ; Theoretical Chemistry: Electronic Structure, Dynamics, Simulation
• Term: from 2013 to 2019
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 226972182

• Final Report Year: 2019

Final Report Abstract

In this research project, we fully elucidated the molecular workings of an extraordinary enzyme, namely “human guanylate binding protein 1” (hGBP1), using large-scale (hybrid QM/MM metadynamics) computer simulations. This GTPase cleaves successively two phosphate bonds of guanosine triphosphate (GTP), thus leading to the respective diphosphate (GDP) and finally to the monophosphate (GMP) species. This is unusual since other small G-proteins, such as Ras, do not fully hydrolyse GTP to GMP, but rather stop after the first step. In particular, our simulations disclose in detail that hGBP1 catalyzes GTP hydrolysis through a complex proton relay pathway with an extended and dynamical hydrogen bonding network involving the nucleophilic water, Ser73, Glu99, GTP, as well as a particular water molecule. The architecture of the active site of hGBP1 is found to be optimally designed to facilitate this proton shuttling and a “composite base” consisting of Ser73, Glu99, a bridging water molecule, and GTP was found to activate the nucleophilic water, thus disclosing an unexpectedly complex nature of the “general base” functionality in the case of hGBP1. GTP was found to be the ultimate proton acceptor, suggesting an “indirect SAC” (substrate-assisted catalysis) mechanism for hGBP1, which is in keeping with the mechanism proposed for the Ras GTPase. The subsequent GDP hydrolysis in hGBP1 was also found to follow a proton relay mechanism in which Glu99 activates the nucleophilic water utilizing two intervening water molecules in a wire-like arrangement without involving Ser73. Moreover, a two-fold reduction in the activation free energy barrier was observed in the enzyme as a result of comparing one-to-one the hydrolysis of GTP in hGBP1 with the corresponding non-enzymatic hydrolysis reaction of a GTP mimic, namely methyl triphosphate, in bulk water serving as the reference system. The significant enzymatic acceleration is traced back to specific hydrogen bond interactions present in the active site of hGBP1 that eventually stabilize the transition state, which is not possible in the reference system. The transition state structure of GDP hydrolysis and free energy barriers obtained from our studies are in excellent agreement with the available experimental results, thus corroborating further our mechanistic findings. Our study further disclosed that GDP sliding within the active site is not limited to hGBP1 but also feasible in other GT- Pases, which in turn, indicate that GDP sliding within the enzyme’s active site cannot be the rate limiting factor for GDP hydrolysis. The other interesting observation we obtained from our study is that eventhough both, Ser73 and Glu99 have been identified to be mechanistically crucial for hGBP1 to carry out GTP hydrolysis, their absence do not halt the GTP hydrolysis in mutant hGBP1s. The alanine substitutions in these two residues found to lead the GTP hydrolysis to alternative reaction routs that have very similar barriers as the wild type. These hitherto unknown reaction channels are established by mechanistically involving far-distant residues using “floating” water molecules, which connect them via hydrogen-bonding bridges to the nucleophilic water molecule, thus allowing for efficient long-distance proton transfer via the Grotthuss mechanism. Given the generic nature of the disclosed detour mechanism that provides the molecular underpinning of what we call “catalytic versatility” and thus mutational robustness of hGBP1, it is expected that the same concept is broadly operational for GTPases.

Publications

• Mechanistic Insights into the Hydrolysis of a Nucleoside Triphosphate Model in Neutral and Acidic Solution, J. Am. Chem. Soc. 134, 6995–7000 (2012); with 18 pages of Supporting Information
  R. Glaves, G. Mathias, and D. Marx
  
• “In Silico” Phosphate Transfer: From Bulk Water to Enzymes, inSiDE (Innovatives Supercomputing in Deutschland) 10(1), 40–43 (2012)
  R. Glaves, G. Mathias, and D. Marx
  
• The GTPase hGBP1 converts GTP to GMP in two steps via proton shuttle mechanisms, Chem. Sci. 8, 371–380 (2017); with 28 pages of Electronic Supplementary Material (ESI)
  R. Tripathi, R. Glaves, and D. Marx
  
• Exposing catalytic versatility of GTPases: taking reaction detours in mutants of hGBP1 enzyme without additional energetic cost, Phys. Chem. Chem. Phys. 21, 859–867 (2019); with 12 pages of Electronic Supplementary Material (ESI)
  R. Tripathi, J. Noetzel, and D. Marx