Probes of ligand-induced conformational change in aspartate aminotransferase.
Sodium borohydride and sodium cyanoborohydride were assessed as potential reagents for determining ligand-induced changes in accessibility to the active-site of aspartate aminotransferase. Rates of reduction of the imine formed between Lys258 and pyridoxal phosphate were determined in the presence of increasing concentrations of the dicarboxylate substrate analogues glutarate and maleate. The rate of reduction decreased to a limiting value which was about 40-fold lower than the equivalent rate in the absence of dicarboxylate. Analysis of the reaction was complicated by the increasing protonation of the imine which accompanied binding of dicarboxylates. Allowing for this increase, the true decrease in accessibility to NaBH3CN was estimated to be approximately 400-fold. Arguments are presented in support of a proposal that the ratio of closed to open conformer of the dicarboxylate-liganded enzyme is approximately 150. The effects of increasing ligand concentration on the reactivity of Cys390 were found to take place in the same range as was observed for NaBH3CN reduction. Conversely, very much higher concentrations of the dicarboxylates were required to protect against proteolysis by trypsin. It is concluded that NaBH3CN reduction and reactivity of cysteine are good determinants of the conformational status of the enzyme but that resistance to tryptic digestion is due to an additional binding mode for the dicarboxylates.
Garnier
A
A
Department of Biochemistry, University of Wales College of Cardiff.
John
R A
RA
D000818:Animals; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D001894:Borohydrides / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D005977:Glutarates; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008298:Maleates; D015335:Molecular Probes; D009206:Myocardium / Q000201:enzymology; D010084:Oxidation-Reduction; D011487:Protein Conformation; D013552:Swine
D001894:Borohydrides; D005977:Glutarates; D008024:Ligands; D008298:Maleates; D015335:Molecular Probes; C025364:sodium borohydride; C030272:maleic acid; C009282:sodium cyanoborohydride; D001219:Aspartate Aminotransferases; C035736:glutaric acid; D003545:Cysteine