title: Location of the redox-active thiols of ribonucleotide reductase: sequence similarity between the Escherichia coli and Lactobacillus leichmannii enzymes.
abstract: The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1-14C]iodoacetamide. The dithiothreitol-reduced E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14C. Sequencing of tryptic peptides shows that 2.8 equiv of 14C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14C. Sequencing of tryptic peptides shows that 1.4 equiv of 14C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I.
authors.affiliation: Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
authors.forename: A N
authors.initials: AN
authors.lastname: Lin
authors.forename: G W
authors.initials: GW
authors.lastname: Ashley
authors.forename: J
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology*; D007460:Iodoacetamide / Q000378:metabolism; D007778:Lactobacillus / Q000201:enzymology*; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D013045:Species Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
chemical_list: D013438:Sulfhydryl Compounds; D012264:Ribonucleotide Reductases; D007460:Iodoacetamide