Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
The mechanism-based inactivation of glutamate decarboxylase (GAD) of E. coli Sukhareva, B. S. & Braunstein, A. E. (1971) Mol. Biol. 5, 302 and of cytosolic aspartate aminotransferase (AAT) by L-serine-O-sulfate John, R. A. & Fasella, P. (1969) Biochem. 8, 4477 has been reinvestigated. The 336 nm form of GAD (1) releases a low molecular weight yellow compound 2 at high pH Likos, J. J. & Metzler, D. E. (1976) Fed. Proc. 35, 1545. The identical compound is released from AAT. Compound 2 is a known compound synthesized by an aldol condensation between pyridoxal phosphate (PLP) and pyruvate Schnackerz, K. D. et al. (1979) Biochem. 18, 3557. The identity of 2 was recognized by Likos private communication. The characterization of 2 isolated from GAD and AAT and comparison with synthetic 2 by 300 MHz NMR spectroscopy is described. The structure of 1 is proposed in collaboration with Likos as an adduct formed by attack of the (beta)-carbon of (alpha)-aminoacrylate on the "internal" Schiff base of PLP with a lysine side chain from the protein. Aminoacrylate is generated from serine sulfate by enzyme-catalyzed (beta)-elimination. Compound 2 is presumably released by attack of hydroxyl ion on 1. Release is prevented by treatment with NaBH(,4) which reduces the C=N or C=O linkage in 1 thereby lowers the acidity of the (beta)-proton removed in the formation of 2. A new "protein factor" required for the inactivation of GAD was discovered. Its partial purification and properties are described. The formation of 2 indicates that GAD does not require decarboxylation during the inactivation with serine sulfate. As described in the reaction with chloroalanine Morino, Y. et al. (1974) J. Biol. Chem. 249, 6684, AAT is inactivated over a 10 min period and the absorption maximum at pH 5.4 shifts from 430 nm to 336 nm (1a). The peak shifts again over a period of 20 h to 455 nm (1b), a behavior entirely similar to that reported by Morino et al. for chloroalanine in the presence of 3 M formate. When the 20 h product 1b is reduced with NaBH(,4) and then heated in a boiling water bath, a material identical to a known reduction product of 2 is released. Therefore, the 20 h product 1b consists of 2 bound to the enzyme. Pathways for the formation of the various compounds are proposed. These findings require a reevaluation of the mechanisms of action of many mechanism-based inactivators of PLP-dependent enzymes.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Ueno, Hiroshi, "Mechanism-based inactivation of glutamate decarboxylase and aspartate aminotransferase with L-serine-O-sulfate " (1982). Retrospective Theses and Dissertations. 7486.