TY - JOUR
T1 - Regulation of aspartate amino-transferase isozymes by glyceraldehyde-3-phosphate
AU - Nisselbaum, Jerome S.
AU - Sweetman, Lawrence
AU - Kopelovich, Levy
N1 - Funding Information:
In order to elucidate further the role of aspartate aminotransferase in carbohydrate metabolism, the activities of the isozymes were investigated under conditions of energy excess, i.e. glucose and fructose feeding. Preliminary observations indicated that when fasted rats, which have elevated levels of the liver anionic isozyme, were fed fructose, the activity of this isozyme returned within 6 hr toward the levels present in the fed animal. This change did not occur when glucose was fed. The refeeding of either sugar did not affect the level of the activity of the cationic isozyme (14). These observations led to an investigation of the effects on purified aspartate aminotransferase isozymes of metabolites which are either unique to fructose metabolism (15) or have been shown to change significantly in rat liver upon * This work was supported in part by grants from the National Cancer Institute (CA-08748)a nd The American Cancer Society (T-431L). 273
PY - 1972
Y1 - 1972
N2 - 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 (Ki = 0.98 mm) and oxaloacetate (Ki not measurable), and completely noncompetitive with respect to l-aspartate (Ki = 0.084 mm) and l-glutamate (Ki = 0.11 mm). Inhibition of the anionic isozyme was mixed partially competitive-partially noncompetitive with respect to α-ketoglutarate (Ki = 1.9 mm) and oxaloacetate (Ki = 1.5 mm) and partially noncompetitive with respect to l-aspartate (Ki = 0.39 mm) and l-glutamate (Ki = 0.57 mm). 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, Ki = 3.08 mm and 1.4 mm for the anionic and cationic isozymes, respectively, and was completely noncompetitive with respect to l-aspartate, Ki = 0.334 mm and 0.135 mm 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 Ki values were 0.77 mm and 1.01 mm 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{lunate}-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{lunate}-amino lysyl group which would react with the inhibitor. Evidence for a Schiff base was obtained by NaBH4 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.
AB - 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 (Ki = 0.98 mm) and oxaloacetate (Ki not measurable), and completely noncompetitive with respect to l-aspartate (Ki = 0.084 mm) and l-glutamate (Ki = 0.11 mm). Inhibition of the anionic isozyme was mixed partially competitive-partially noncompetitive with respect to α-ketoglutarate (Ki = 1.9 mm) and oxaloacetate (Ki = 1.5 mm) and partially noncompetitive with respect to l-aspartate (Ki = 0.39 mm) and l-glutamate (Ki = 0.57 mm). 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, Ki = 3.08 mm and 1.4 mm for the anionic and cationic isozymes, respectively, and was completely noncompetitive with respect to l-aspartate, Ki = 0.334 mm and 0.135 mm 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 Ki values were 0.77 mm and 1.01 mm 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{lunate}-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{lunate}-amino lysyl group which would react with the inhibitor. Evidence for a Schiff base was obtained by NaBH4 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.
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U2 - 10.1016/0065-2571(72)90018-0
DO - 10.1016/0065-2571(72)90018-0
M3 - Article
C2 - 4653824
AN - SCOPUS:0015457957
SN - 0065-2571
VL - 10
SP - 273
EP - 287
JO - Advances in Enzyme Regulation
JF - Advances in Enzyme Regulation
IS - C
ER -