Conformation change lactate dehydrogenase

ISOTOPE TRAPPING STICKY SUBSTRATES Substrate-induced conformational change, INDUCED FIT MODEL SUBSTRATE INHIBITION ABORTIVE COMPLEX FORMATION LACTATE DEHYDROGENASE LEE-WILSON EQUATION... 

A key structural and mechanistic feature of lactate and malate dehydrogenases is the active site loop, residues 98-110 of the lactate enzyme, which was seen in the crystal structure to close over the reagents in the ternary complex.49,50 The loop has two functions it carries Arg-109, which helps to stabilize the transition state during hydride transfer and contacts around 101-103 are the main determinants of specificity. Tryptophan residues were placed in various parts of lactate dehydrogenase to monitor conformational changes during catalysis.54,59,60 Loop closure is the slowest of the motions. 

The specificities of the enzymes are also nicely explained The enantiomers of the substrates of L-lactate and D-glyceraldehyde 3-phosphate dehydrogenases cannot be productively bound the hydrophobic pocket of alcohol dehydrogenase will not bind the charged side chains of lactate etc. However, we do not know if conformational changes occur during catalysis or if there is strain. 

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... 

The raison d etre for an ordered mechanism may be that binding of the coenzyme creates the binding site for the second substrate by inducing a conformational change of the enzyme 23). There is direct evidence for this from recent X-ray diffraction studies of lactate dehydrogenase 24). The conformational change will, however, be manifest kinetically only if it occurs as an elementary step separate from coenzyme binding, thus. 

Tarmy EM, Kaplan NO. (1968). Kinetics of Escherichia coli B D-lactate dehydrogenase and evidence for pyruvate controlled change in conformation. J Biol Chem, 243, 2587-2596. 

Although, favorable factors are present, the system prefers to remain aromatic. Hence, the formation of NADH in the enzymatic system could be driven by conformational changes that shift the equilibrium toward the nonaromatic species. However, in 1978, a German group (276) observed an intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase using a coenzyme-substrate covalent analogue composed of lactate and NAD+. 

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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