13C-NMR and transient kinetic studies on lactate dehydrogenase [Cys(13CN)165]. Direct measurement of a rate-limiting rearrangement in protein structure.

Chemical modification of cysteine-165 in pig heart lactate dehydrogenase to produce lactate dehydrogenase [Cys(13CN)165] introduces an covalently bound, enriched 13C probe at a position adjacent to the active cen. The signal from the thiocyanate probe is clearly visible at 47 ppm relative to dioxane. On formation of binary complexes with NAD+ and NADH, no signal change is detected. Formation of the ternary complexes E-NADH-oxamate and E-NAD+-oxalate results in an upfield shift of the signal of 1.2 ppm. These results interpreted as demonstrating that binding of the substrate analogue induces a conformational change a position adjacent to the active centre. Exchange experiments in which the enzyme is poised in dynamic equilibrium between binary and ternary complexes show that the rate at which the probe senses a change environment is the same as the kinetically observed unimolecular event which limits the enzyme-catalyst reduction of pyruvate. The two processes show the same dependence on temperature, solvent composition and pH. These results indicate that the rate-limiting isomerisation corresponds to a rearrangement of the protein in the region of cysteine-165.