Contrasting Values of Commitment Factors Measured from Viscosity, pH, and Kinetic Isotope Effects: Evidence for Slow Conformational Changes in theD-Amino Acid Oxidase Reaction

Abstract

The flavoproteinD-amino acid oxidase catalyzes the oxidation ofD-amino acids to imino acids. Previous studies of pH and isotope effets on the reaction withD-alanine as substrate have established that the enzyme substrate complex partitions forward toward carbon–hydrogen bond cleavage 10 times as fast as the amino acid dissociates and that overall turnover is limited by product release. However, theV/Kvalue forD-alanine is not affected by the solution viscosity, while theVmaxvalue is only 15% limited by diffusion. These results are interpreted as evidence for a protein conformational change between an open conformation which binds substrates and a closed complex within which catalysis occurs. Such a model is supported by the recently reported structure of the enzyme–benzoate complex (A. Mattevi, M. A. Vanoni, F. Todone, M. Rizzi, A. Teplyakov, A. Coda, M. Bolognesi, and B. Curti (1996)Proc. Natl. Acad. Sci. USA93,7496–7501).