Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225→serine) by its substrate CDP

S. S. Mao, M. I. Johnston, J. M. Bollinger, J. Stubbe

Research output: Contribution to journalArticlepeer-review

36 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1485-1489
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume86
Issue number5
DOIs
StatePublished - 1989

All Science Journal Classification (ASJC) codes

  • General

Fingerprint Dive into the research topics of 'Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225→serine) by its substrate CDP'. Together they form a unique fingerprint.

Cite this