Location of the redox-active thiols of ribonucleotide reductase: sequences similarity between the Escherichia coli and Lactobacillus leichmannii enzymes
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), Vol. 26:22
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CARBON 14 COMPOUNDS
TRACER TECHNIQUES
OXIDOREDUCTASES
AMINO ACID SEQUENCE
TRITIUM COMPOUNDS
ACETAMIDE
ESCHERICHIA COLI
LACTOBACILLUS
LIQUID COLUMN CHROMATOGRAPHY
NUCLEOTIDES
SCINTILLATION COUNTING
SUBSTRATES
THIOLS
AMIDES
BACTERIA
CHROMATOGRAPHY
COUNTING TECHNIQUES
ENZYMES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MICROORGANISMS
MOLECULAR STRUCTURE
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
SEPARATION PROCESSES
550201* - Biochemistry- Tracer Techniques