Mechanism-based inhibition of ribonucleoside diphosphate reductase from Corynebacterium nephridii by...

The interaction of the adenosylcobalamin-dependent ribonucleoside diphosphate reductase of Corynebacterium nephridii with 2'-C-methyladenosine diphosphate (2'-C-methylADP) has been investigated in more detail [Ong, S. P., McFarlan, S. C., & Hogenkamp, H. P. C. (1993) Biochemistry 32, 11397-11404]. This nucleotide analog partitioned between normal reduction to 2'-deoxy-2'-C-methyladenosine diphosphate and decomposition to adenine, 2-methylene-3(2H)-4-methylfuranone, and presumably pyrophosphate. Reaction of the reduced enzyme with 2'-C-methylADP caused the development of a chromophore at 318 nm that is characteristic of the modification of the enzyme by the furanone [Harris, G., Ator, M., & Stubbe, J. (1984) Biochemistry 23, 5214-5225]. Incubation of [5'-3H2]-2'-C-methylADP with reduced reductase resulted in the covalent incorporation of the radiolabel into the protein and into aquocobalamin. A similar incubation of the enzyme, the labeled nucleotide analog, and dithiothreitol resulted in the formation of three radioactive hydrophilic compounds. Mass spectroscopic analysis of one of these compounds showed the presence of 2-methylene-3(2H)-4-methylfuranone. 2'-Deoxy-2'-C-methylADP is a very effective promoter of the tritium exchange reaction between [5'-3H2]adenosylcobalamin and the solvent, confirming that the exchange reaction is an integral part of the overall reduction. All these observations are consistent with the proposal that 2'-C-methylADP serves as a substrate and a mechanism-based inhibitor of the ribonucleotide reductase of C. nephridii, indicating that the enzyme is able to catalyze the conversion of the nucleotide analog to a 2'-deoxy-2'-C-methyl-3'-ketonucleotide that can collapse to the reactive 2-methylene-3(2H)-4-methylfuranone. Surprisingly, 2'-C-methylADP did not serve as either a substrate or an inhibitor of the ribonucleoside diphosphate reductase of Escherichia coli.