The Active Center of Aspartate Transaminase: A FLUORINE-19 NUCLEAR MAGNETIC RESONANCE STUDY OF THE ANION BINDING SITE

Abstract

Abstract The binding of trifluoroacetate anion to aspartate transaminase (EC 2.6.1.1) has been measured by the changes of the chemical shift in the 19F nuclear magnetic resonance spectrum. The dissociation constants for the enzyme-trifluoroacetate complex are pH dependent with values ranging from 15 to 36 mm at pH values between 5 and 8. Stoichiometry shows that there is only one anion binding site. The pH-dependence of trifluoroacetate binding indicates that a group in the enzyme with a pKa of 6.2 ± 0.2 is responsible for anion binding. Apoenzyme and holoenzyme after reduction of the pyridoxyl-lysine aldimine with NaBH4 (reduced enzyme), show the same pH dependence pattern and the same pKa value for trifluoroacetate binding as native enzyme. Controlled photooxidation of the active site histidine residue results in the elimination of the anion binding site. Chloride and succinate compete with trifluoroacetate for the same site in the native, apoenzyme, and reduced enzymes. On the other hand, the pseudosubstrate α-methylaspartate, which binds covalently to the enzyme to form the initial aldimine enzyme-substrate complex, competes with trifluoroacetate for the binding site only in the native enzyme, but this does not occur after the removal or reduction of the active site Schiff's base, i.e. in the apoenzyme and reduced enzyme. The positively charged anion binding site in the protein is postulated to be the active site histidine. A model is proposed for mutual exclusion between anion and substrate from the active center without a sharing of common subsites. The model is consistent with the stoichiometry, competition effects and pH dependence of ligand binding, as well as with the elimination of anion binding after photooxidation of the active center histidine without a subsequent effect on substrate affinity.