Location of the redox-active thiols of ribonucleotide reductase: sequences similarity between the Escherichia coli and Lactobacillus leichmannii enzymes

Journal Article · Tue Nov 03 04:00:00 EST 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00396a006· OSTI ID:5403375

Lin, A N.I.; Ashley, G W; Stubbe, J

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-/sup 14/C)iodoacetamide. The dithiothreitol-reduce E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of /sup 14/C. Sequencing of tryptic peptides shows that 2.8 equiv of /sup 14/C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of /sup 14/C 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 /sup 14/C. Sequencing of tryptic peptides shows that 1.4 equiv of /sup 14/C 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.

• Research Organization: Univ. of Wisconsin, Madison
• OSTI ID: 5403375
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:22; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETAMIDE
AMIDES
AMINO ACID SEQUENCE
BACTERIA
CARBON 14 COMPOUNDS
CHROMATOGRAPHY
COUNTING TECHNIQUES
ENZYMES
ESCHERICHIA COLI
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LACTOBACILLUS
LIQUID COLUMN CHROMATOGRAPHY
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
SCINTILLATION COUNTING
SEPARATION PROCESSES
SUBSTRATES
THIOLS
TRACER TECHNIQUES
TRITIUM COMPOUNDS