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.
Mao
S S
SS
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Johnston
M I
MI
Bollinger
J M
JM
Stubbe
J
J
D003001:Cloning, Molecular; D003545:Cysteine; D003565:Cytidine Diphosphate / Q000494:pharmacology*; D003597:Cytosine Nucleotides / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D009154:Mutation; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000235:genetics; D012694:Serine
D003597:Cytosine Nucleotides; D012694:Serine; D003565:Cytidine Diphosphate; D012264:Ribonucleotide Reductases; D003545:Cysteine