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Volume 272, Number 50, Issue of December 12, 1997 pp. 31533-31541

A New Mechanism-based Radical Intermediate in a Mutant R1 Protein Affecting the Catalytically Essential Glu⁴⁴¹ in Escherichia coli Ribonucleotide Reductase

(Received for publication, July 24, 1997, and in revised form, September 12, 1997)

Annika L. Persson , Mathias Eriksson ^§ , Bettina Katterle , Stephan Pötsch , Margareta Sahlin and Britt-Marie Sjöberg

From the Departments of  Molecular Biology and  Biophysics, Stockholm University, S-106 91 Stockholm and the ^§ Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, S-75149 Uppsala, Sweden

The invariant active site residue Glu⁴⁴¹ in protein R1 of ribonucleotide reductase from Escherichia coli has been engineered to alanine, aspartic acid, and glutamic acid. Each mutant protein was structurally and enzymatically characterized. Glu⁴⁴¹ contributes to substrate binding, and a carboxylate side chain at position 441 is essential for catalysis. The most intriguing results are the suicidal mechanism-based reaction intermediates observed when R1 E441Q is incubated with protein R2 and natural substrates (CDP and GDP). In a consecutive reaction sequence, we observe at least three clearly discernible steps: (i) a rapid decay (k₁  1.2 s¹) of the catalytically essential tyrosyl radical of protein R2 concomitant with formation of an early transient radical intermediate species, (ii) a slower decay (k₂ = 0.03 s¹) of the early intermediate concomitant with formation of another intermediate with a triplet EPR signal, and (iii) decay (k₃ = 0.004 s¹) of the latter concomitant with formation of a characteristic substrate degradation product. The characteristics of the triplet EPR signal are compatible with a substrate radical intermediate (most likely localized at the 3-position of the ribose moiety of the substrate nucleotide) postulated to occur in the wild type reaction mechanism as well.

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