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J. Biol. Chem., Vol. 279, Issue 30, 31050-31057, July 23, 2004

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Enhancement by Effectors and Substrate Nucleotides of R1-R2 Interactions in Escherichia coli Class Ia Ribonucleotide Reductase^*

Alex Kasrayan, Pernilla Larsson Birgander, Lucia Pappalardo, Karin Regnström, MariAnn Westman, Agneta Slaby, Euan Gordon¶, and Britt-Marie Sjöberg||

From the Department of Molecular Biology & Functional Genomics, Stockholm University, SE-10691 Stockholm, Sweden

Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric _{2 2} structure. The component ₂called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the ₂ component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2–3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.

Received for publication, January 21, 2004 , and in revised form, May 10, 2004.

^* This work was supported in part by grants from the Swedish Cancer Foundation and Grant 98-0207 from the EU-TMR. The Biacore 3000 instrument used for some of the studies was financed generously by a grant from the Swedish Research Council, and the Biacore 1000 instruments used in the initial studies were generous gifts of BioVitrum AB (Stockholm, Sweden). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Istituto di Chimica Biomolecolare del CNR, Comprensorio Olivetti, Edificio 70, Via Campi Flegrei 34, I-80078 Pozzuoli (Napoli), Italy.

Present address: Dept. of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, 372 Fairfield Way, Storrs, CT 06269.

^¶ Dept. of Molecular Biotechnology, Chalmers University of Technology, P. O. Box 462, SE-40530 Gothenburg, Sweden.

^|| To whom correspondence should be addressed. Tel.: 46-8-164150; Fax: 46-8-166488; E-mail: britt-marie.sjoberg{at}molbio.su.se.

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