Conformational changes of aspartate aminotransferases in the region of Cys-45 residue observed by means of spin label

Abstract

Two thiol groups located at the surface of the aspartate aminotransferase molecule ( l-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) can be alkylated by a paramagnetic label, viz. 2,2,6,6-tetramethylpiperidine-1-oxyl-4-iodoacetate. The modified enzyme restores the activity almost completely. Under the conditions used the spin label is not bound by any other functional groups of the aspartate aminotransferase molecule. Binding of ligands (substrates and quasisubstrates) at the enzyme active site induces changes of the EPR spectrum, which are reversible: removal of ligands by gel filtration through a Sephadex G-25 column results in the disappearance of these changes. One of the accessible thiol groups (Group I) was selectively alkylated by maleic acid whereas the other one (Group II) was labeled with the spin label. In this case addition of ligands did not affect the mobility of the spin label. It can be concluded, therefore, that it is only the spin label bound to the Group I that is sensitive to the interaction of a ligand with the active site. This group in the native enzyme was labeled with [ 14C]maleic acid. Chromatography of the chymotrypsin digest of the treated protein yielded radioactive peptide; thereafter its partial structure was identified. The amino acid sequence of the peptide corresponds to that of the enzyme region from 41 to 48 (Trp residue in the peptide under study was destroyed during acid hydrolysis). Previously it has been shown that the residue Tyr-40 is located in the region of the enzyme active site. The localization of the cysteine under study (Cys-45) makes it possible to think that this amino acid residue is rather close to the active site of the aspartate aminotransferase. However, the distance ( >17 A ̊ ) between this residue and the active site as seen from the data obtained is not sufficient to provide spacial overlapping between the spin label and the Schiff's base of coenzyme with the substrate molecule. Therefore, the effect of substrate upon the mobility of the spin label is mediated by conformational changes in a certain region of the active site.