Free Energy Surface of the Michaelis Complex of Lactate Dehydrogenase: A Network Analysis of Microsecond Simulations

Abstract

It has long been recognized that the structure of a protein creates a hierarchy of conformations interconverting on multiple time scales. The conformational heterogeneity of the Michaelis complex is of particular interest in the context of enzymatic catalysis in which the reactant is usually represented by a single conformation of the enzyme/substrate complex. Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of two forms of the cofactor nicotinamide adenine dinucleotide (NADH and NAD+). Recent experimental results suggest that multiple substates exist within the Michaelis complex of LDH, and they show a strong variance in their propensity toward the on-enzyme chemical step. In this study, microsecond-scale all-atom molecular dynamics simulations were performed on LDH to explore the free energy landscape of the Michaelis complex, and network analysis was used to characterize the distribution of the conformations. Our results provide a detailed view of the kinetic network of the Michaelis complex and the structures of the substates at atomistic scales. They also shed light on the complete picture of the catalytic mechanism of LDH.

Conformational Heterogeneity in the Michaelis Complex of Lactate Dehydrogenase: An Analysis of Vibrational Spectroscopy Using Markov and Hidden Markov Models

“…Recently, we estimated the free energy surface of the Michaelis complex of LDH using the cut-based free energy profile (FEP) method. 22 Even though different substrate binding modes were resolved, the basins on the FEP and the substates resolved by vibrational spectroscopy were only compared indirectly through hydrogen bond analysis. Due to the limitation of the method, the relaxation time scales of the system were underestimated by about 4 orders of magnitude.…”

Computational Design of Synthetic Enzymes

“…Their mutant is expected to lower the free energy barrier of the reaction, hence exhibits higher acceleration rate compared to the natural enzyme 139. Focusing on correlated motions with the active site loop, Pan and Schwartz explained how the strong conformational heterogeneity may open alternative mechanisms for the chemical step that need to be accounted for when designing de novo enzymes 137. They extended their analysis to designed Kemp Eliminases and their improved variants under LDE, for which they found that LDE selected mutations favor a more dynamic active site that makes use of the donoracceptor compression 140.…”

Mechanistic Analysis of Fluorescence Quenching of Reduced Nicotinamide Adenine Dinucleotide by Oxamate in Lactate Dehydrogenase Ternary Complexes

“…But due to its weaker interaction, the contribution would be less important. Recently, molecular dynamics study on pyruvate ternary complex also suggested that the hydrogen bonds between these residues and pyruvate form in the encounter complex . Following structural rearrangements and the loop closure, the active site becomes tighter and hydrogen bonding, electrostatic interactions and the hydrophobic effect all come to be stronger.…”