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

Abstract

Lactate dehydrogenase (LDH) catalyzes the interconversion of pyruvate and lactate. Recent isotope-edited IR spectroscopy suggests that conformational heterogeneity exists within the Michaelis complex of LDH, and this heterogeneity affects the propensity toward the on-enzyme chemical step for each Michaelis substate. By combining molecular dynamics simulations with Markov and hidden Markov models, we obtained a detailed kinetic network of the substates of the Michaelis complex of LDH. The ensemble-average electric fields exerted onto the vibrational probe were calculated to provide a direct comparison with the vibrational spectroscopy. Structural features of the Michaelis substates were also analyzed on atomistic scales. Our work not only clearly demonstrates the conformational heterogeneity in the Michaelis complex of LDH and its coupling to the reactivities of the substates, but it also suggests a methodology to simultaneously resolve kinetics and structures on atomistic scales, which can be directly compared with the vibrational spectroscopy.

Triple Isotope Effects Support Concerted Hydride and Proton Transfer and Promoting Vibrations in Human Heart Lactate Dehydrogenase

“…The LDH enzyme demonstrates instructive catalysis–dynamics relationships 6,8,10,20–31 and plays an important role in metabolism. 36,47–49 We optimized the expression and purification methods to produce human heart LDH at a high yield, which may be adapted to other LDH enzymes to facilitate research with this enzyme family.…”

“…6,8,10,20–31 LDH catalyzes the final step of anaerobic glycolysis, the reduction of pyruvate to L-lactate using reduced nicotinamide adenine dinucleotide (NADH) as the hydride donor. This reaction is reversible with an equilibrium strongly favoring the reduction of pyruvate to l -lactate.…”

Promoting Vibrations and the Function of Enzymes. Emerging Theoretical and Experimental Convergence

“…These developments required computational definition of the transition state parameters. , Slower protein motions that lead to catalytically productive complexes were also characterized (in a range of promoting vibrations). , Further refinements of this work using Markov state models have strengthened this approach. , Other important developments support the promoting vibration concept. First, we showed that in lactate dehydrogenase, the promoting vibration is a preferred thermal channel; i.e., the enzyme takes thermal energy and transmits it along this channel .…”

Electric Fields and Enzyme Catalysis

“…In contrast, data from our laboratory accrued over the past few years have supported the view that most vibrational frequency variation (in carbonyls, at least) can be explained through a static electric field–difference dipole effect (53, 91), not by bond weakening or strain (i.e., changes in the force constant) (103, 104). This represents a departure from how vibrational measurements (both infrared and Raman) have traditionally been interpreted in enzymology (105–107), and efforts are ongoing to reinterpret this corpus of biochemical data within this physical framework (108, 109). …”