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.