Reaction coordinate of an enzymatic reaction revealed by transition path sampling

Abstract

The transition path sampling method previously applied in our group to the reaction catalyzed by lactate dehydrogenase was used to generate a transition path ensemble for this reaction. Based on analysis of the reactive trajectories generated, important residues behind the active site were implicated in a compressional motion that brought the donor-acceptor atoms of the hydride closer together. In addition, residues behind the active site were implicated in a relaxational motion, locking the substrate in product formation. Although this suggested that the compressionrelaxation motions of these residues were important to catalysis, it remained unproven. In this work, we used committor distribution analysis to show that these motions are integral components of the reaction coordinate.protein dynamics ͉ catalysis T he relationship between enzymatic structure and function remains an area of intense research. Many studies have demonstrated that factors such as electrostatic and entropic effects are significant to enzyme function (1). The role of dynamics in catalysis, however, is less well understood. Protein motions such as conformational fluctuations on the millisecond timescale have been shown to influence substrate binding and the height of the barrier to reaction. The role of vibrational motions on the femtosecond to picosecond timescale and their connection to catalysis remain controversial. It has been suggested that the vibrational motions that exist within the protein are in equilibrium with the reaction and thermally averaged along the reaction coordinate (2, 3). But we and others have suggested that vibrational motions within the protein may in fact be coupled to the reaction coordinate (4). Previous work in our group has suggested the coupling of these vibrational motions to the reaction coordinate and has developed methods to study their effect on enzyme reactions (5).The search for these subpicosecond motions (''protein promoting vibrations'') was first applied to the enzyme horse liver alcohol dehydrogenase. This work suggested there existed motions, protein-promoting vibrations (PPVs), that coupled directly to the reaction coordinate and that were on the timescale of barrier crossing (6). The algorithm was then tested on another enzyme system lactate dehydrogenase (LDH), and similar PPVs were identified that, along with conformational fluctuations, helped explain the preference of the heart isoform to produce pyruvate and of muscle isoform to produce lactate (7).Further investigation into the mechanism of LDH was achieved with the transition path sampling (TPS) method (8). TPS is a computational method that can simulate rare events in complex systems. Using a Monte Carlo approach, a reactive path ensemble connecting reactants to products can be defined without prior knowledge of the reaction coordinate. This method allows mechanistic details to be identified from reactive paths generated with no bias (9). A reactive path ensemble was generated for LDH, and it was found that, in all reactive trajectori...

A two-step nucleotide-flipping mechanism enables kinetic discrimination of DNA lesions by AGT

“…Transition path sampling (TPS) has transformed the computational study of rare events in molecular systems over the last decade (11,12), but its application to complex, biological systems has been limited (24,25,(27)(28)(29). Efficient means for generating initial paths (14) together with informatic methods (13) that we introduced now enable us to harvest a statistically significant number of trajectories of a biomedically important stochastic process in its entirety and identify the features that characterize the ensemble of transition states.…”

Transition Path Sampling Study of the Reaction Catalyzed by Purine Nucleoside Phosphorylase

“…The results suggested both concerted and stepwise mechanisms of hydride and proton transfer as possible paths, and showed that the hydride transfer was facilitated by motions of residues far from the active site. A subsequent study by our group [8] determined a reaction coordinate that incorporates these residue motions.…”

Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase

“…Slower (millisecond) motions are involved in conformational changes that occur during substrate binding and product release (1)(2)(3)(4), whereas femtosecond motions (promoting vibrations) are proposed by computational studies to be involved in the chemical step of transition-state formation (covalent bond changes) (5)(6)(7)(8). The role of fast (femtosecond) enzyme dynamics in the catalytic cycle has remained elusive and controversial due to the experimental challenge of probing femtosecond motions in enzymes.…”