Non-Enzymatic Glycosylation (or Glycation) and Inhibition of the Pig Heart Cytosolic Aspartate Aminotransferase by Glyceraldehyde 3-Phosphate

Abstract

Glyceraldehyde 3-phosphate (Glyc3P), a glycolytic intermediate, non-enzymatically glycosylated (or glycated) and inhibited the pig heart cytoplasmic aspartate aminotransferase (cAAT). Glyc3P (5.0 mM) decreased cAAT activity by 47% after 1 min at 23 degrees C. cAAT activity remained unchanged after a 24 h incubation with either glucose 6-phosphate (5.0 mM) or ribose 5-phosphate (5.0 mM). Increasing the incubation pH from 6.4 to 7.8 or the incubation temperature from 23 degrees C to 50 degrees C enhanced Glyc3P's inhibitory effect on cAAT activity. Glyc3P (250-500 µM) decreased the thermal stability of cAAT as evidenced by lowering the T(m) or temperature that caused a 50% irreversible loss of cAAT activity (69 degrees C, control; 58.5 degrees C, 500 µM Glyc3P). Glyc3P decreased cAAT amino group content and increased glycation products, which were measured by adduct formation, fluorescence and protein crosslinking.

Glycation of Aspartate Aminotransferase and Conformational Flexibility

“…Protein glycation causes crosslinking (10,11), changes proteolytic susceptibility (12), increases transition metal binding (13), increases protein fluorescence (5), and alters signal transduction mechanisms (18). Glycation of proteins alter protein function (5), and glycation of small nitrogenous compounds yields fluorescent compounds (3), alter nutritive value (19) and produce genotoxic compounds (20). Reactivity of amino acids (21) and glycating agents (8) differ considerably.…”

“…Glyceraldehyde 3-phosphate, a glycolytic intermediate, glycates (2) the cardiac cytosolic isoform and inhibits its activity (5). Additionally, the rat liver cytosolic AAT is susceptible in vivo glycation (7).…”

“…cAAT resists thermal denaturation presumably due to its arrangement of ␣-helices (4). Despite its structural stability, cAAT is readily modified by glycation (nonenzymatic glycosylation) (5).…”

Acrolein Modifies and Inhibits Cytosolic Aspartate Aminotransferase

“…10 Incubated samples were diluted in a 100 mM sodium phosphate buffer ðpH ¼ 7:4Þ containing 0.1 mM pyridoxal 5-phosphate (PLP) (final assay PLP concentration was 1 mM). Aliquots (0.2 mg cAAT) were assayed at 378C in an 80 mM sodium phosphate buffer ðpH ¼ 7:4Þ containing 200 mM L-aspartate, 12 mM a-ketoglutarate, 0.2 mM NADH and 1.1 I.U.…”

Multiple Binding Partners

“…Glyceraldehyde 3-phosphate rapidly reacts and inactivates enzymes [21]. The trioses are more reactive than the cyclic pentoses or hexoses that have their carbonyl groups masked in a hemiacetal linkage.…”