Active Site Structural Features for Chemically Modified Forms of Rhodanese

Abstract

In the course of the reaction catalyzed by rhodanese, the enzyme cycles between two catalytic intermediates, the sulfur-free and the sulfur-substituted (persulfide-containing) forms. The crystal structure of sulfur-free rhodanese, which was prepared in solution and then crystallized, is highly similar to that of sulfur-substituted enzyme. The inactivation of sulfur-free rhodanese with a small molar excess of hydrogen peroxide relies essentially on a modification limited to the active site, consisting of the oxidation of the essential sulfhydryl to sulfenyl group (-S-OH). Upon reaction of the sulfur-free enzyme with monoiodoacetate in the crystal, the Cys-247 side chain with the bound carboxymethyl group is forced into a conformation that allows favorable interactions of the carboxylate with the four peptide NH groups that participate in hydrogen bonding interactions with the transferable sulfur atom of the persulfide group in the sulfur-substituted rhodanese. It is concluded that active site-specific chemical modifications of sulfur-free rhodanese do not lead to significant changes of the protein structure, consistent with a high degree of similarity of the structures of the sulfur-free and sulfur-substituted forms of the enzyme both in solution and in the crystal.

Enzymatic Activity of the Arabidopsis Sulfurtransferase Resides in the C-Terminal Domain But Is Boosted by the N-Terminal Domain and the Linker Peptide in the Full- Length Enzyme

“…A closer look at the amino acids which correspond to the replaced cysteine residues in Arabidopsis ST1 in the three-dimensional model of bovine rhodanese (Gliubich et al, 1996) shows that C294 and C304 are probably located at the surface of the protein molecule, C152 could be close to the surface. C332 and C339 are buried in the molecule in close neighbourhood to each other, however, probably without direct interaction.…”

“…Site-directed mutagenesis studies of bovine rhodanese have shown that out of four conserved and reduced cysteines, C247 is the only cysteine required for the activity of the enzyme (Miller-Martini et al, 1994a). A persulfide link between this essential sulfhydryl group and the transferred sulfur is formed in the ES intermediate (Ploegman et al, 1979;Gliubich et al, 1996).…”

S-Sulfhydration of the Catalytic Cysteine in the Rhodanese Domain of YgaP is Complex Dynamic Process

“…It has been stipulated that the S-sulfhydrated enzyme undergoes a significant conformational change. However, the crystal structures of the sulfur-free as well as S-sulfhydrated enzyme are very similar [9]. Even more puzzling results came from the study that investigated in detail the impact of S-sulfhydration on the structure and dynamics of the rhodanese domain of the E. coli integral membrane protein YgaP using solution NMR as the main technique [10].…”

Haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 refined at 1.15 Å resolution

“…Furthermore, a difference density peak >20' was observed at a distance of 1.7 A Ê from Cys150 SG, which was preliminarily interpreted as a covalently linked O atom. Therefore, residue 150 was included in the model as an S-hydroxycystine, as has been observed previously in rhodanese (Gliubich et al, 1996). Solvent sites which had no 2mF o À DF c electron density above 2' were removed.…”