Cytoplasmic Aspartate Aminotransferase: Syncatalytic Sulfhydryl Group Modification

Abstract

Cytoplasmic aspartate aminotransferase possesses five free sulfhydryl groups per monomer. In the native enzyme two thiol groups (groups I and II) appear to be positioned on the exterior of the molecule as reflected by their reactivity toward sulfhydryl reagents. Modification of these two groups does not impair catalytic activity; blocking by N-ethylmaleimide even increases the activity to 125%. One further thiol group (group III) reacts at a rate five orders of magnitude slower than either one of the exterior thiol groups. The remaining sulfhydryl groups (groups IV and V) are nonreactive in the native enzyme. The reactivity of thiol group III changes within two orders of magnitude during the course of catalysis. The reactivity is lowest in the free pyridoxal enzyme and is slightly increased in the presence of the substrate analogs α-methyl aspartate or erythro-β-hydroxyaspartate which form either the aldimine or the semiquinoide intermediates, respectively. Maximal reactivity is attained in the presence of the substrate pair glutamate and α-ketoglutarate, when an equilibrium of all covalent enzyme-substrate intermediates is established. Thus, thiol group III appears to be most reactive in the ketimine intermediate. The alterations in reactivity from intermediate to intermediate are interpreted to reflect conformational changes of the enzyme-coenzyme-substrate complex occurring syncatalytically, i.e. during catalysis. Modifications of thiol group III with N-ethylmaleimide, 5,5′-dithiobis(2-nitrobenzoate), or tetranitromethane reduce aminotransferase activity to less than 5% of the initial value. The derivative modified with 5,5′-dithiobis(2-nitrobenzoate) is fully reactivated by deblocking of thiol group III. Enzymatic activity is also markedly reduced in mixed disulfide derivatives between thiol group III and glutathione, sulfite, mercaptoethanol, or methyl mercaptan. In contrast, the enzyme derivative modified with cyanide was found to be 60% enzymatically active. Apparently, bulky or charged substituents on thiol group III impair the catalytic activity, whereas modification with the small and uncharged S-cyano group is compatible with virtually full transaminase activity. Thus, sulfhydryl group III seems not to have an essential role in either catalysis or maintenance of an active enzyme conformation. The specificity of chemical modifications of the different classes of thiol groups was confirmed by analysis of the CNBr peptides. From a CNBr fragment of the enzyme derivative modified with cyanide at thiol group III the tryptic peptide containing the labeled residue was isolated. Its sequence (Cys, Gly, Leu, Thr)Thr-Lys indicates that thiol group III is located in the COOH-terminal portion of tryptic peptide 22 reported by Ovchinnikov et al. ((1971) Fed. Eur. Biochem. Soc. Lett. 17, 133).