Degree Type


Date of Award


Degree Name

Doctor of Philosophy


Biochemistry, Biophysics and Molecular Biology


N-methylpyridoxal 5'-phosphate (N-MePLP) was synthesized by treatment of vitamin B(,6) phosphate with dimethylsulfate (Pfeuffer et al., 1972). N-methylpyridoxamine 5'-phosphate (N-MePMP) was prepared by non-enzymatic transamination of N-MePLP with glutamate;Electronic absorption spectra of individual ionic forms of highly purified N-MePLP were evaluated together with its pK(,a) values. The formation constants, acid dissociation constants and absorption spectra of various ionic forms of the Schiff base of N-MePLP with valine have been determined. The equilibrium constant for the nonenzymatic transamination between N-MePLP and glutamate was estimated;The apo form of cytosolic aspartate aminotransferase (AAT) from pig heart reconstituted with N-MePLP (E.N-MePLP) was crystallized. Polarized light absorption spectra of a single crystal were obtained. Absorption spectra of individual ionic forms of E.N-MePLP in solution were also reported;The binding of N-MePLP and of N-MePMP to the apo form of AAT were examined. The pK observed for the bound aldehydic form of the coenzyme analog was determined. Dissociation constants for chloride, (alpha)-ketoglutarate, glutarate, succinate and adipate in the anion complexes with E.N-MePLP were measured. The reversible reaction of E.N-MePLP with L-glutamate was studied in detail. Estimations were made of equilibrium constants for various steps in the overall transamination process. The reasons for both the low coenzyme activity of N-MePLP and the distinct change in pK observed for E.N-MePLP compared with that for the native enzyme were discussed;O-methylpyridoxamine 5'-phosphate (O-MePMP) was synthesized according to Besedina et al. (1971). It was oxidized to O-methypyridoxal 5'-phosphate (O-MePLP) according to the methods of Pocker & Fischer (1969);The apo form of AAT reconstituted with O-MePLP (E.O-MePLP) was also crystallized. Although possessing no detectable activity, E.O-MePLP reacts with L-asparate to produce transiently the quinonoid intermediate (Furbish et al., 1969; Mora et al., 1972). Upon further examination, the turnover of the quinonoid intermediate was found to be pH sensitive. A pK was estimated as 8.1. The mechanism of the reaction was discussed;References;Besedina, T. I; Breuson, Yu. N; Karpeiskii, M. Ya; Stambolieva, N. A; Turchin, K. F; Florent'ev, V. L. Khim. Getero. Soed. 1971, 7, 368;Furbish, F. S; Fonda, M. L; Metzler, D. E. Biochemistry 1969, 8, 5169;Mora S; Bocharov, A. L; Ivonov, V. I; Karpeisky, M. Ya; Mamaeva,O. R; Stambolieva, N. A. Molec. Biol. (SSSR) 1972, 6, 113;Pocker, A; Fischer, D. H. Biochemistry 1969, 8, 5181.



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Victor John Chen



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283 pages

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Biochemistry Commons