Characterization of the substrate binding dynamics of lactate dehydrogenase.

摘要

We examine here the dynamics of forming the Michaelis complex of the enzyme lactate dehydrogenase (LDH) by characterizing the binding kinetics and thermodynamics of oxamate (a substrate mimic) to the binary LDH/NADH complex over multiple time scales, from nanoseconds to tens of milliseconds. In order to access such a wide time range, we employ standard stopped-flow kinetic approaches (slower than 1 ms) and laser induced temperature-jump relaxation spectroscopy (10 ns - 10 ms). The emission from the nicotinamide ring of NADH, and the IR absorption of the carbonyl stretch mode of oxamate (bound to the binary complex) at 1606 cm-1 are used as markers of structural transformations. The results are well explained by a kinetic model that has binding taking place via a sequence of steps: the formation of an encounter complex in a bi-molecular step followed by two unimolecular transformations on the microsecond/millisecond time scales. All steps are well described by single exponential kinetics. It appears that the various key components of the catalytically competent architecture are brought together as separate events, with the formation of strong hydrogen bonding between active site His195 and substrate early in binding and the closure of the catalytically necessary protein surface loop over the bound substrate as the final event of the binding process. This loop remains closed during the entire period that chemistry takes place for native substrates; however, motions of other key molecular groups bringing the complex in and out of catalytic competence appear to occur on faster times scales. The on-enzyme Kd's, (the ratios of the microscopic rate constants for each unimolecular step), are not far from one. Either substantial, ca. 10-15%, transient melting of the protein or rearrangements of hydrogen bonding and solvent interactions of a number of water molecules or both appears to take place to permit substrate access to the protein binding site. The nature of activating the various steps in the binding process seems to be one overall involving substantial entropic changes.