Dehydrogenases conformational changes

Yeast Alcohol Dehydrogenase. Conformational changes are known to play an important role in the mechanism of enzyme action. The existence of conformational changes in enzymes bound to coenzymes or substrates has been postulated by several investigators (Nozaki et al., 1957 Sekuzu et al., 1957 Yonetani and Theorell, 1962 DiSabato and Kaplan, 1964 DiSabato and Kaplan, 1965). Hvidt and her... 

W. R. Laws and J. D. Shore, Spectral evidence for tyrosine ionization linked to a conformational change in liver alcohol dehydrogenase ternary complexes, J. Biol. Chem. 254, 2582-2584 (1979). 

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

Ordered mechanisms often occur in the reactions of the NAD+-linked dehydrogenases, with the coenzyme binding first. The molecular explanation for this is that the binding of the dinucleotide causes a conformational change that increases the affinity of the enzyme for the other substrate (see Chapter 16). 

Clearly, this method cannot be applied to systems in which there are irreversible chemical processes. It is most suitable for situations involving simple ligand binding (such as NAD+ with a dehydrogenase), inhibitor binding, or conformational changes in the protein. There have been some attempts to combine the temperature-jump with the stopped-flow method. 

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... 

When trying to determine the 3D structure of binary and ternary complexes of Drosophila alcohol dehydrogenase (DADH), researchers initial attempts to soak apo-form crystals with the oxidized coenzyme (NAD+) failed. The crystals cracked after several hours and became unusable. This suggested that the coenzyme, upon binding to the enzyme, induced a conformational change that seriously affected the crystal packing. The same phenomenon prevented solution of the HL-ADH 3D structure for many years. 

Branden, C.-l., Ekiund, H. Coenzyme-induc conformational changes and substrate binding in liver alcohol dehydrogenase in Molecular Interactions and Activity in Proteins, Ciba Foundation Symp. 60 (New Kries), pp. 63-80, Ezceipta M Uca, Amsterdam, 1978... 

How eould this eatalytie bias be controlled One possibility is that the proton transfer pathway eould eontribute to specifieity (Peters et al., 1998). Another possibility is that differences in midpoint potential of the FeS clusters (or other redox sites) that constitute the intramolecular wire could be tuned to facilitate one of the two directions of the reaction. For example, these redox sites could best match the midpoint potentials of a particular oxidized or reduced electron carrier (Holm and Sander, 1999). Apparently, a conformational change in succinate dehydrogenase, coupled to the reduction of FAD, is responsible for its catalytic bias for fumarate reduction (Hirst et al., 1996). 

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