Direct Evidence of Catalytic Heterogeneity in Lactate Dehydrogenase by Temperature Jump Infrared Spectroscopy

Abstract

Protein conformational heterogeneity and dynamics are known to play an important role in enzyme catalysis, but their influence has been difficult to observe directly. We have studied the effects of heterogeneity in the catalytic reaction of pig heart lactate dehydrogenase using isotope edited infrared spectroscopy, laser-induced temperature jump relaxation, and kinetic modeling. The isotope edited infrared spectrum reveals the presence of multiple reactive conformations of pyruvate bound to the enzyme, with three major reactive populations having substrate C2 carbonyl stretches at 1686, 1679, and 1674 cm−1, respectively. The temperature jump relaxation measurements and kinetic modeling indicate that these substates form a heterogeneous branched reaction pathway, and each substate catalyzes the conversion of pyruvate to lactate with a different rate. Furthermore, the rate of hydride transfer is inversely correlated with the frequency of the C2 carbonyl stretch (the rate increases as the frequency decreases), consistent with the relationship between the frequency of this mode and the polarization of the bond, which determines its reactivity toward hydride transfer. The enzyme does not appear to be optimized to use the fastest pathway preferentially but rather accesses multiple pathways in a search process that often selects slower ones. These results provide further support for a dynamic view of enzyme catalysis where the role of the enzyme is not just to bring reactants together but also to guide the conformational search for chemically competent interactions.

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

“…3 In our model shown here, the two binding modes could correspond to the two well-defined substates, basins A and B, while the transition regions could correspond to the inhomogeneous state, basin C. However, in contrast to the experimental results, 3 our model shows that basins A and B can interconvert either directly or by going through a transition region.…”

“…Recent experiments have shown that conformational heterogeneity exists in the Michaelis complex of LDH, 1–3 and the propensity toward the hydride transfer reaction may vary widely among the different substates of the Michaelis complex. However, the relationship between the substates is still elusive, and the detailed structural features of the substates are unclear.…”

Mechanism of Thermal Adaptation in the Lactate Dehydrogenases

“…In studies of the phLDH·NADH·pyruvate complex, a small IR band representative of the encounter complex species is in fact observed. 11,13 …”

“…Indeed, it has been shown that the frequency of the C 2 =O stretch is a quantitative monitor of propensity toward catalysis. 11–13 Laser-induced temperature jump spectroscopy, employing isotope edited IR and fluorescence monitors of evolving structure, has yielded the kinetic pathway from ligand binding to catalysis. 13–16 These studies have led to an unparalleled identification of mechanistically related structure and dynamics within the Michaelis complex of LDH.…”

Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact

“…6B). Variability in free energy barriers caused by multiple molecular states has precedent in studies of ligand binding and catalysis, where differences between protein conformational substates and their relative stabilities are thought to cause variations in the activation energy of different pathways (53)(54)(55)(56)(57)(58). The idea of multiple parallel routes for a single reaction is analogous to the welldocumented concept of multiple protein folding pathways (55,56,59).…”