Substrate Inhibition of D-Amino Acid Transaminase and Protection by Salts and by Reduced Nicotinamide Adenine Dinucleotide: Isolation and Initial Characterization of a Pyridoxo
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- 作者：Peter W. van Ophem ; Shawn D. Erickson ; Alvaro Martinez del Pozo ; Ivan Haller ; Brian T. Chait ; Tohru Yoshimura ; Kenji Soda ; Dagmar Ringe ; Gregory Petsko ; and James M. Manning
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：March 3, 1998
- 年：1998
- 卷：37
- 期：9
- 页码：2879 - 2888
- 全文大小：164K
- 年卷期：v.37,no.9(March 3, 1998)
- ISSN：1520-4995
文摘
D-Amino acid transaminase, a pyridoxalphosphate (PLP) enzyme, is inactivated by its naturalsubstrate, D-alanine, concomitant with its
-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., andManning, J. M. (1993) J. Biol.Chem. 268, 17687-17694]. 
-Decarboxylation ofD-aspartate to D-alanine leads also to thisinactivation[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 ahigh-performance liquid chromatography-based methodfor the determination of pyridoxo cofactors, we detected a newintermediate closely related to theinactivation by D-alanine; its formation occurred at thesame rate as the inactivation and upon reactivationit reverted to PLP. Conditions were found under which it wascharacterized by ultraviolet-visible spectralanalysis and mass spectroscopy; it is a pyridoxamine phosphate-likecompound with a C2 fragment derivedfrom the substrate attached to the C'-4 of the pyridinium ring and ithas a molecular mass of 306 consistentwith this structure. In the presence of D-serine, slowaccumulation of a quinonoid intermediate is alsorelated to inactivation. The inactivation can be prevented bysalts, which possibly stabilize the protonatedaldimine coenzyme complex. The reduced cofactor, nicotinamideadenine dinucleotide, preventsD-aspartate-induced inactivation. Both of these eventsalso are related to formation of the novel intermediate.
-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., andManning, J. M. (1993) J. Biol.Chem. 268, 17687-17694]. -Decarboxylation ofD-aspartate to D-alanine leads also to thisinactivation[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 ahigh-performance liquid chromatography-based methodfor the determination of pyridoxo cofactors, we detected a newintermediate closely related to theinactivation by D-alanine; its formation occurred at thesame rate as the inactivation and upon reactivationit reverted to PLP. Conditions were found under which it wascharacterized by ultraviolet-visible spectralanalysis and mass spectroscopy; it is a pyridoxamine phosphate-likecompound with a C2 fragment derivedfrom the substrate attached to the C'-4 of the pyridinium ring and ithas a molecular mass of 306 consistentwith this structure. In the presence of D-serine, slowaccumulation of a quinonoid intermediate is alsorelated to inactivation. The inactivation can be prevented bysalts, which possibly stabilize the protonatedaldimine coenzyme complex. The reduced cofactor, nicotinamideadenine dinucleotide, preventsD-aspartate-induced inactivation. Both of these eventsalso are related to formation of the novel intermediate.