Influence of Ligands on the Coenzyme Dissociation Constants in Octopine Dehydrogenase

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Abstract
Various ligands influence the affinity of coenzymes to octopine dehydrogenase. When the enzyme is saturated by the alternative substrate l‐arginine plus pyruvate or d‐octopine, the dissociation constants of NAD and NADH decrease more than one order of magnitude. Studies with stereoisomeric substrate analogues show that the configuration of the asymmetric carbon atom of the arginine moiety has a profound influence on the binding, but changing the configuration of the alanine moiety does not prevent good binding. Changes in pH and ionic strength do not appreciably alter the dissociation constants of NAD and NADH, which is taken as an indication that electrostatic forces are not very important for the binding process and that the driving forces for the coenzyme binding are essentially hydrophobic in nature. The values of the coenzyme dissociation constants in the presence of various ligands are consistent with a mechanism of compulsory order for coenzyme binding and release. The binding of nicotinamide 1:N6‐ethanoadenine dinucleotide (ɛ‐NAD, an analog of NAD with a fluorescent tag on the adenine ring) is accompanied by a remarkable enhancement of the coenzyme fluorescence and by a blue shift of the fluorescent maximum, showing that the adenosine moiety must be directly involved in the coenzyme binding, also that the coenzyme probably binds in an open conformation to the enzyme. The binding data obtained by following the enhancement of the ɛ‐NAD fluorescence are in agreement with those obtained by monitoring the quenching of the protein fluorescence.

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