Lactate dehydrogenase catalytic conformational changes

Alcohol oxidation requires release of a proton, which formally comes from the alcohol. In other dehydrogenases such as lactate dehydrogenase, proton release occurs simultaneously with hydride transfer. In liver ADH proton release can be demonstrated, by reaction of the proton with an indicator such as thymol blue or phenol red in stopped-flow spectrophotometry, to be faster than hydride transfer, 270 vs. 150 s and unaffected by use of deuterated substrate, so it occurs before hydride transfer. Binding of the NAD+ nicotinamide ring is accompanied by a conformational change of ADH bringing the catalytic zinc about 0.1 nm closer to the... 

This enzyme requires ordered addition of its substrates - the nucleotide has to bind prior to lactate or pyruvate. Similarly the nucleotide dissociates after the other substrate. The conformation change to form the reactive complex occurs when both NADH and pyruvate are bound. In the reverse direction, when the concentration of free pyruvate is negligible, an isomerization step (E E) has to occur after pyruvate has dissociated, but before NADH can dissociate. Therefore an additional step is involved in NADH dissociation after catalytic turnover. Some evidence for two step binding of NADH to lactate dehydrogenase has been found by Wu etal. 99 ) even in the absence of pyruvate. Similar phenomena are observed during the dissociation of the products after ATP hydrolysis by myosin (see section 5.1). Some of these events may still be subject to revision, but it is clear that product dissociation from enzymes requires quite detailed analysis. Some of the approaches to this problem have been outlined in section 5.2. 

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