Control of d-octopine formation in scallop adductor muscle as revealed through thermodynamic studies of octopine dehydrogenase

SUMMARY Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus (Linné, 1758), catalyses the NADH-dependent condensation of l-arginine and pyruvate to d-octopine, NAD+ and water during escape swimming and subsequent recovery. During exercise, ATP is mainly provided by the transphosphorylation of phospho-l-arginine and to some extent by anaerobic glycolysis. NADH resulting from the glycolytic oxidation of 3-phosphoglyceraldehyde to 1,3-bisphosphoglycerate is reoxidized during d-octopine formation. In some scallops d-octopine starts to accumulate during prolonged, strong muscular work, whereas in other species d-octopine formation commences towards the end of swimming and continues to rise during subsequent recovery. The activity of OcDH is regulated by a mandatory, consecutive mode of substrate binding in the order NADH, l-arginine and pyruvate, as demonstrated by isothermal titration calorimetry. The first regulatory step in the forward reaction comprises the binding of NADH to OcDH with a dissociation constant Kd of 0.014±0.006 mmol l–1, which reflects a high affinity and tight association of the apoenzyme with the co-substrate. In the reverse direction, NAD+ binds first with a Kd of 0.20±0.004 mmol l–1 followed by d-octopine. The binary OcDH–NADH complex associates with l-arginine with a Kd of 5.5±0.05 mmol l–1. Only this ternary complex combines with pyruvate, with an estimated Kd of approximately 0.8 mmol l–1 as deduced from pyruvate concentrations determined in the muscle of exhausted scallops. At tissue concentrations of pyruvate between 0.5 and 1.2 mmol l–1 in the valve adductor muscle of fatigued P. maximus, binding of pyruvate to OcDH plays the most decisive role in initiating OcDH activity and, therefore, in controlling the onset of d-octopine formation.