Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225----serine) by its substrate CDP.
The B1 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1) has been overexpressed using the pT7-5/pGP1-2 system developed by Tabor and Richardson [Tabor, S. & Richardson, C. (1985) Proc. Natl. Acad. Sci. USA 82, 1074-1078]. This method has allowed the preparation of two mutant B1 subunits in which two of the four thiols postulated to be within the active site of the enzyme, Cys-225 and Cys-759, have been changed to serines. Incubation of the [Ser225]B1 mutant with the B2 subunit, [U-14C]CDP, and the allosteric effector ATP results in production of cytosine, destruction of the tyrosyl radical in B2, radiolabeling of the protein, and cleavage of the B1 subunit into two pieces of 26 and 61.5 kDa. This process is independent of the identity of reductant. The [Ser759]B1 mutant reduces CDP in the presence of thioredoxin/thioredoxin reductase at 7.7% the rate of wild-type B1. When dithiothreitol is utilized as reductant, however, the rate of CDP reduction with [Ser759]B1 is identical to that observed with wild type.
21 references, click to expand
-
A rapid assay for CDP reductase activity in mammalian cell extracts.Anal Biochem. 1970 Mar;34:123-30 PMID: 5440901
-
ENZYMATIC SYNTHESIS OF DEOXYRIBONUCLEOTIDES.V. PURIFICATION AND PROPERTIES OF THIOREDOXIN REDUCTASE FROM ESCHERICHIA COLI B.J Biol Chem. 1964 Oct;239:3445-52 PMID: 14245401
-
A colony bank containing synthetic Col El hybrid plasmids representative of the entire E. coli genome.Cell. 1976 Sep;9(1):91-9 PMID: 788919
-
Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase.J Biol Chem. 1977 May 10;252(9):2891-9 PMID: 16006
-
Ribonucleoside diphosphate reductase from Escherichia coli. An immunological assay and a novel purification from an overproducing strain lysogenic for phage lambdadnrd.J Biol Chem. 1977 Sep 10;252(17):6132-8 PMID: 197082
-
Purification of thioredoxin, thioredoxin reductase, and glutathione reductase by affinity chromatography.J Biol Chem. 1977 Sep 25;252(18):6367-72 PMID: 330529
-
DNA sequencing with chain-terminating inhibitors.Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463-7 PMID: 271968
-
Ribonucleotide reductase--a radical enzyme.Science. 1983 Aug 5;221(4610):514-9 PMID: 6306767
-
Mechanism of inactivation of Escherichia coli and Lactobacillus leichmannii ribonucleotide reductases by 2'-chloro-2'-deoxynucleotides: evidence for generation of 2-methylene-3(2H)-furanone.Biochemistry. 1984 Oct 23;23(22):5214-25 PMID: 6391538
-
A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes.Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074-8 PMID: 3156376
-
The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.Nucleic Acids Res. 1985 Dec 20;13(24):8765-85 PMID: 3001650
-
Mechanism of inactivation of Escherichia coli ribonucleotide reductase by 2'-chloro-2'-deoxyuridine 5'-diphosphate: evidence for generation of a 2'-deoxy-3'-ketonucleotide via a net 1,2 hydrogen shift.Biochemistry. 1985 Dec 3;24(25):7214-21 PMID: 3910098
-
Cloning, overproduction, and purification of the B2 subunit of ribonucleoside-diphosphate reductase.J Bacteriol. 1986 Feb;165(2):363-6 PMID: 3511029
-
Identification of the stable free radical tyrosine residue in ribonucleotide reductase.EMBO J. 1986 Aug;5(8):2037-40 PMID: 3019680
-
Current ideas on the chemical mechanism of ribonucleotide reductases.Pharmacol Ther. 1985;30(3):301-29 PMID: 3916287
-
Products of the inactivation of ribonucleoside diphosphate reductase from Escherichia coli with 2'-azido-2'-deoxyuridine 5'-diphosphate.Biochemistry. 1987 Jun 16;26(12):3408-16 PMID: 3307907
-
Location of the redox-active thiols of ribonucleotide reductase: sequence similarity between the Escherichia coli and Lactobacillus leichmannii enzymes.Biochemistry. 1987 Nov 3;26(22):6905-9 PMID: 3322391
-
Structure-function studies of the large subunit of ribonucleotide reductase from Escherichia coli.Biochem Soc Trans. 1988 Apr;16(2):91-4 PMID: 3286319
-
Comparative amino acid sequence of fructose-1,6-bisphosphatases: identification of a region unique to the light-regulated chloroplast enzyme.Proc Natl Acad Sci U S A. 1988 Aug;85(15):5379-83 PMID: 2840657
-
Total sequence, flanking regions, and transcripts of bacteriophage T4 nrdA gene, coding for alpha chain of ribonucleoside diphosphate reductase.J Biol Chem. 1988 Nov 5;263(31):16242-51 PMID: 2846540
-
Reaction mechanism of ribonucleoside diphosphate reductase from Escherichia coli. Oxidation-reduction-active disulfides in the B1 subunit.J Biol Chem. 1974 Aug 10;249(15):4858-62 PMID: 4152559
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