Regulation of aspartate amino-transferase isozymes by glyceraldehyde-3-phosphate

Abstract

This review concerns our work on glyceraldehyde-3-P as a time-dependent inhibitor of rat liver aspartate aminotransferase isozymes, and the possibility that glyceraldehyde-3-P may function as a rapidly acting regulator of aspartate aminotransferase. The d-isomer and the dl-racemate of glyceraldehyde-3-P were equally effective for each isozyme. Study of several glycolytic intermediates indicated that the conjoint presence of the free aldehyde and the phosphoryl residue was necessary for inhibition. Maximum inhibition of the anionic isozyme occurred at pH values from 8.4 to 10.3; the cationic isozyme was optimally inhibited at pH 7.4. Keto acid substrates decreased the inhibition, whereas amino acid substrates accentuated it. Inhibition of the cationic isozyme was completely competitive with respect to α-ketoglutarate ( K i = 0.98 m m ) and oxaloacetate ( K i not measurable), and completely noncompetitive with respect to l-aspartate ( K i = 0.084 m m ) and l-glutamate ( K i = 0.11 m m ). Inhibition of the anionic isozyme was mixed partially competitive-partially noncompetitive with respect to α-ketoglutarate ( K i = 1.9 m m ) and oxaloacetate ( K i = 1.5 m m ) and partially noncompetitive with respect to l-aspartate ( K i = 0.39 m m ) and l-glutamate ( K i = 0.57 m m ). These data suggest that both keto acids bind to the isozymes at a single site and compete with glyceraldehyde-3-P for that site, whereas the amino acids bind to a site other than the one for which keto acids and glyceraldehyde-3-P compete. Homologues of glyceraldehyde-3-P were also investigated. Ribose-5-P, fructose-6-P and glucose-6-P did not inhibit either isozyme since they exist as the internal hemiacetals. d-erythrose-4-P was a time-dependent inhibitor of both isozymes. Inhibition was completely competitive with respect to α-ketoglutarate, K i = 3.08 m m and 1.4 m m for the anionic and cationic isozymes, respectively, and was completely noncompetitive with respect to l-aspartate, K i = 0.334 m m and 0.135 m m for the anionic and cationic isozymes, respectively. Inhibition by glycolaldehyde-P was not time-dependent and was completely competitive with respect to α-ketoglutarate and uncompetitive with respect to l-aspartate for both isozymes. The K i values were 0.77 m m and 1.01 m m for the anionic and cationic isozymes, respectively. We propose that glyceraldehyde-3-P and its homologues inhibit aspartate aminotransferase isozymes by forming a Schiff base with one of the ϵ-amino groups of lysine at the enzymically active site. Aspartate would potentiate inhibition by converting the enzyme to the pyridoxamine form, thereby exposing a second ϵ-amino lysyl group which would react with the inhibitor. Evidence for a Schiff base was obtained by NaBH 4 reduction of apoisozymes in the presence of dl-glyceraldehyde-3-P. This prevented restoration of activity upon addition of pyridoxal-5′-P. It seems likely that the divalent phosphoryl group on the inhibitor molecule is involved in the competition at the keto acid binding site. Our results suggest the possibility that glyceraldehyde-3-P may be implicated in the regulation, in vivo, of gluconeogenesis as well as other metabolic pathways by affecting the activity of the isozymes of aspartate aminotransferase.