SUBSTRATE INHIBITION OF D AMINO ACID TRANSAMINASE & PROTECTION BY SALTS

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D-amino acid transaminase, a pyridoxal phosphate enzyme, is inactivated by its natural substrate, D-alanine, concomitant with it's ? -decarboxylation [Martinez del Pozo A., Yoshimura, T. Bhatia, M.B. Futaki, S. Manning, J.M., Ringe, D., and Soda K. (1992) Biochemistry 31, 6018-6023; Bhatia, M.B., Martinez del Pozo, A., Ringe, D., Yoshimura, T., Soda, K. and Manning, J.M. (1993) J. Biol. Chem. 268, 17687-17694]. ?-Decarboxylation of D-aspartate to D-alanine leads also to this inactivation [Jones, W.M., van Ophem, P.W., Pospischil, M.A., Ringe, D., Petsko, G., Soda, K., and Manning, J.M. (1996) Prot. Sci. 5, 2545-2551]. Using an HPLC-based method for the determination of pyridoxo-cofactors, we detected a new intermediate closely related to the inactivation by D-alanine; its formation occurred at the same rate as the inactivation and upon reactivation by D-alanine; its formation occurred at the same rate the inactivation and upon reactivation it reverted to PLP. C onditions were found under which it was characterzed by UV-vis spectral analysis and mass spectroscopy; it is a pyridoxamine phosphate-like compound with a C2-fragment derived from the substrate attached to the C'-4 of the pyridinum ring and it has a molecular mass of 306 consistent with this structure. In the presence of D-serine, slow accumulation of a quinonoid intermediate is also related to inactivation. The inactivation can be prevented by salts, which possibly stabilize the protonated aldimine coenzyme complex. The reduced co-factor, NADH, prevents D-aspartate induced inactivation. Both of these events also are related to formation of the novel intermediate.