Substrate inhibition of D-amino acid transaminase and protection by salts and by reduced nicotinamide adenine dinucleotide: isolation and initial characterization of a pyridoxo intermediate related to inactivation.
D-Amino acid transaminase, a pyridoxal phosphate (PLP) enzyme, is inactivated by its natural substrate, D-alanine, concomitant with its alpha-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]. beta-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) Protein Sci. 5, 2545-2551]. Using a high-performance liquid chromatography-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 it reverted to PLP. Conditions were found under which it was characterized by ultraviolet-visible 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 pyridinium 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 cofactor, nicotinamide adenine dinucleotide, prevents D-aspartate-induced inactivation. Both of these events also are related to formation of the novel intermediate.
van Ophem
P W
PW
Northeastern University, Boston, Massachusetts 02115, USA.
Erickson
S D
SD
Martinez del Pozo
A
A
Haller
I
I
Chait
B T
BT
Yoshimura
T
T
Soda
K
K
Ringe
D
D
Petsko
G
G
Manning
J M
JM
D000409:Alanine / Q000494:pharmacology; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D051263:D-Alanine Transaminase; D004791:Enzyme Inhibitors / Q000494:pharmacology; D009243:NAD / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D011733:Pyridoxamine / Q000031:analogs & derivatives / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D012492:Salts; D012694:Serine / Q000494:pharmacology; D000637:Transaminases / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D012492:Salts; D009243:NAD; D012694:Serine; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D019289:Pyruvic Acid; D000637:Transaminases; D051263:D-Alanine Transaminase; D000409:Alanine; C010627:pyridoxamine phosphate; C024593:pyridoxine 5-phosphate