Dissociation constants, dehydrogenases

The FAD-requiring enzymes in mammalian systems include the D- and L-amino acid oxidases, mono- and diamine oxidases, glucose oxidase, succinate dehydrogenase, a-glycerophosphate dehydrogenase, and glutathione reductase. FMN is a cofactor for renal L-amino acid oxidase, NADH reductase, and a-hydroxy acid oxidase. In succinate dehydrogenase, FAD is linked to a histidyl residue in liver mitochondrial monoamine oxidase, to a cysteinyl residue. In other cases, the attachment is nonco-valent but the dissociation constant is very low. 

Colorimetric and fluorimetric NH3 sensors contain mixtures of pH indicators having suitable dissociation constants at the tip of the fiber bundle. The measuring solution is separated from this indicator layer by an NH3 gas-permeable membrane covered by an immobilized de-aminating enzyme, e.g. urease (Wolfbeis, 1987 Arnold, 1987). The fluorimetric indication of NADH has been used in optical biosensors for lactate, pyruvate, and ethanol, where the respective dehydrogenase is immobilized at the tip of an optical NADH sensor (Arnold, 1987 Wangsa and Arnold, 1988). 

In this mechanism, the K values ( a/ o, b/ o) are dissociation constants for the ternary complex. Equation (8) holds, and in addition 4>AB/A = Keb and pq/0p = Keq- The mechanism was early rejected for beef heart lactate dehydrogenase on the grounds that enzyme-lactate and enzyme-pyruvate compounds having these dissociation constants... 

The conclusion that even with the best substrate, ethanol, dissociation of NAD occurs from the active ternary complex is consistent with the evidence-from isotope exchange experiments 32), mentioned previously, that the dissociation of coenzyme is not greatly suppressed in the ternary complex compared with the binary complex, in contrast to liver alcohol dehydrogenase. This is also indicated by the initial rate data in another way 4>a/4>o for the preferred pathway mechanism approximates to the dissociation constant for NAD from the ternary complex (Table I), and is reasonably constant for the three primary alcohols and approximately equal to the dissociation constant of E-NAD, determined independently 40). 

The main value of product inhibition studies of dehydrogenases has been to distinguish between ordered and random mechanisms and to provide additional kinetic estimates of the dissociation constants of enzyme-coenzyme compounds. On both counts the method has been especially useful for reactions that are essentially irreversible or for other reasons cannot be studied in both directions 122,138). It is also in such circumstances that product inhibition studies are most reliable because, as Alberty (7) emphasized when proposing the method, with readily reversible reactions it may be difficult to estimate true initial rates with small concentrations of substrates in the presence of a product. The reality of ternary complexes in an ordered mechanism of the Theorell-Chance type has also been demonstrated with several enzymes (134) by product inhibition studies. 

Dissociation Constants for Coenzyme Compounds of NAD-Linked Dehydrogenases... 

Dissociation Constants foe Coenzyme Compounds op Rabbit Muscle Glyceraldehyde-3-Phosphate Dehydrogenase... 

There is no uniformity in the effect of pH on the dissociation constants of the NAD compounds. The pronounced decrease of Xe.nad for liver alcohol dehydrogenase from pH 6.0 to pH 10.0 was attributed to ionization of a water molecule coordinated to the active center zinc atom and Coulombic interaction with the nicotinamide N-1 (64)- The absence of a similar pH effect with the other dehydrogenases studied so far is understandable on this basis. Structural studies of the compounds of liver alcohol dehydrogenase with ADPR and 1 10-phenanthroline, competitive... 

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