EXAM OF RIBONUCLEOTIDE REDUCTASE USING UNNATURAL AMINO ACID MUTAGENESIS

University Of Calif-Los Alamos Nat Lab, Los Alamos NM

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Abstract

The SIR provided (I)-Se-Cys; 150mg The ribonucleotide reductase from Lactobacillus leichmannii catalyzed the reduction of NTPs to dNTPs using the cofactor adenosyllcobalamin. Understanding the mechanisms for NTP reduction is important not only because this enzyme is essential for the production of dNTPs and is therefore an important target for drug design, but also because the enzyme is proposed to reduce the NTPs via a novel, radical mechanism. Central to the proposed mechanisms is the formation of an active-site thiyl radical at position Cys 408. To provide evidence for this proposed mechanisms and to understand the role of the thiyl radical in catalysis, this cysteine is being replaced with a variety of amino acids using unnatural amino acid mutagenesis. This methodology allows an amino acid to be synthesized in the lab and then incorporated site-specifically in a protein. Given that the S-H bond strength is similar to that of the substrate C-H bond that is proposed to be homolyzed in the first step of the reduction, Cys 408 is being replaced by amino acids, such as selenocysteine, that are similar in structure to cysteine, but differ in X-H bond strength and kinetic reactivity. Once the amino acids are incorporated into ribonucleotide reductase, the mutant enzymes will be tested for their ability to catalyze the reduction of CTP to dCTP and the exchange of 3H from 3H-adenosyloblamin into 3H2) and may be examined using Electron Spin Resonance Spectroscopy. These replacements should provide insight into the thermodynamic and kinetic requirements for enzyme-catalyzed radical reactions.