CRYSTALLOGRAPHIC AND FLUORESCENCE STUDIES OF LIGAND-BINDING TO N-CARBAMOYLSARCOSINE AMIDOHYDROLASE FROM ARTHROBACTER SP

Crystal structures of N-carbamoylsarcosine amidohydrolase (CSHase; EC 3.5.1.59) have been analyzed by X-ray diffraction methods with two dif ferent inhibitors bound to the active site at 2.28 Angstrom and 2.37 A ngstrom resolution. The catalytic center of the enzyme could be identi fied on the basis of these structures. The four substrate binding site s are situated at the intersubunit interfaces of the compact dimers AB and CD of the homotetrameric enzyme. Both inhibitors inactivate the e nzyme irreversibly through covalent binding of their aldehyde groups t o the thiol group of the active-site cysteine residue Cys177. Within t he identified substrate binding sites a number of residues from differ ent subunits are involved in hydrogen bonding of the inhibitors. Two r esidues (Ala172 and Thr173) that form an unusual cis-peptide bond at t he binding site are important components in fixing the examined inhibi tors by hydrogen bonds. An electrochemical enzyme assay for CSHase was used to test the effect of inhibitors and substrate analogs on the en zyme's activity, revealing the high substrate specificity of CSHase. T he intrinsic tryptophan fluorescence of CSHase increases strongly upon substrate and inhibitor binding. As most of the tryptophyl residues a re located at the active sites, they are thus considerably affected by ligand binding. Fluorescence-detected stopped-flow measurements have been used to study the kinetics of glyoxylate and substrate binding to CSHase. Substrate and inhibitor binding could clearly be distinguishe d in the stopped-flow experiments. Inhibitor binding reveals at least three different elementary processes, whereas substrate binding is muc h faster and contains phases with different signs in amplitude. (C) 19 96 Academic Press Limited