THEORETICAL-STUDY OF THE SUBSTRATE MECHANISM OF RIBONUCLEOTIDE REDUCTASE

The substrate mechanism of ribonucleotide reductase (RNR) is studied u sing a hybrid DFT method (B3LYP) with large basis sets. With models fo r the calculations based on recently determined X-ray structures, diff erent mechanisms of the steps leading from a ribonucleotide to a deoxy ribonucleotide are investigated. It is suggested that the transformati on occurs in six steps. The role of the amino acid residues present at the active site are studied in detail. Three cysteine residues are kn own to be necessary for the formation of a deoxyribonucleotide, but ve ry recently a glutamic acid residue has also been shown to be essentia l. The calculations indicate that this glutamic acid residue, Glu441, is important both in steps 2 and 4.; In the suggested step 2, Glu441 t ransfers a hydrogen from the C3'-OH group to the C2'-OH group, and in step 4 it is proposed to participate in a cyclic transition state brid ging the carbon and oxygen atoms of a keto group at C3'. In both these steps an asparagine, Asn437, also plays an important role in reducing the barrier heights for the reactions. The rate-limiting step of the substrate reactions is suggested to be step 4, where a cysteine residu e attacks the C3' center of the ribose ring. The disulfide bond is pro posed to be formed in step 5. Dielectric. effects from the surrounding protein are very small and of almost no importance in this process.