Aspartate aminotransferase reconstituted with N- and O-methylated vitamin B6 phosphates

Abstract

Eapoapo form of aspartate aminotransferase E.N-MePLP aspartate aminotransferase reconstituted with N-methylpyridoxal 5^-phosphate E.O-MePLP aspartate aminotransferase reconstituted with 0-methylpyridoxal 5'-phosphate kg a-ketoglutarate MDH malic dehydrogenase NADH the reduced form of g-nicotinamide adenine dinucleotide N-MePLP N-methylpyridoxal 5'-phosphate N-MePMP 0-methylpyridoxamine 5'-phosphate nmr nuclear magnetic resonance ORD optical rotary dispersion 0-MePLP 0-methylpyridoxal 5'-phosphate 0-MePMP 0-methylpyridoxamine 5'-phosphate PLP pyridoxal 5'-phosphate PMP pyridoxamine 5'-phosphate TEA.HCl triethanolamine hydrochloride Another attractive aspect of aspartate aminotransferase with respect to the study of coenzyme-protein interaction is the fact that the vitamin Bg cofactor can be removed readily from the protein. In its place, a selection of analogs of pyridoxal 5'-phosphate with various structural modifications can be incorporated. The apoenzyme reconstituted with these analogs displays different degrees of activity. The study of these apoenzyme-analog complexes can provide clues to the mechanisms of inter action between the coenzyme and various functional groups on the protein. Recently, the crystallization of both the mitochondrial and the cytoplasmic isoenzymes has sparked new excitement in the enzymology of transamination (Gehring et al., 1977; Arnone et al., 1977; Borisov et al., 1978). The three-dimensional structures of these proteins are now being determined by X-ray crystallography. The crystalline enzymes have been shown to be catalytically competent. Crystalline enzyme-substrate complexes of natural substrates and various inhibitory ligands as well as crystalline apoenzyme-analog complexes have been prepared (Metzler et al., 1978). Many of these possess linear dichroism and their polarized light absorption spectra have been recorded. Observations of these spectra together with the forthcoming knowledge of the three-dimensional structures of the proteins will provide a means of examining the inter action of the coenzyme with the protein at the atomic level.The study reported in this thesis was undertaken to further characterize two apoenzyme-analog complexes of aspartate aminotransferase.The N-and 0-methylated derivatives of pyridoxal 5'-phosphate have been incorporated into the enzyme (Furbish et al., 1969; Mora et al., 1972). Compounds 23 24 25 26 0.5-4.45 o.gs <2 min^ 'V/10 min^ 322,382,4084 3924 positive at 3804 380,42O4 3774 310(shoulder)4 310(shoulder)4 04 7.5II l4 <2 min to give band at 326 nm4 <2 min4 slow to give 335 nm4 V. s low4 5 min to give a small transient at 485 nm4 5 min, loss of 485 mn4 gave small band at 480 nmll