Affinity labeling of ribonucleotide reductase by the 2',3'-dialdehyde derivatives of ribonucleotides.

Ribonucleotide reductase from Corynebacterium nephridii is rapidly inactivated by the 2',3'-dialdehyde derivatives of CDP (dial-CDP) and ADP (dial-ADP). The analog of CDP causes the progressive inactivation of ribonucleotide reductase activity with Ki of 0.26 mM and a maximum inactivation rate of 0.092 min-1 at saturating concentrations of dial-CDP. The modified enzyme remains inactive even after extensive dialysis. The four common nucleoside diphosphates (ADP, GDP, CDP, and UDP) protect the enzyme against inactivation by dial-CDP. Experiments with [3H]dial-CDP, [14C]dial-ADP, and [32P]dial-ADP demonstrate that the nucleoside moieties of these nucleotide analogs become covalently attached to the enzyme and that inorganic pyrophosphate is eliminated. The stoichiometry of this inactivation, determined with [3H]dial-CDP and [14C]dial-ADP, is 0.6-0.8 site modified per subunit of enzyme. The results suggest that the enzyme catalyzes the elimination of pyrophosphate and that the resulting alpha, beta-unsaturated nucleoside dialdehyde or its corresponding alpha, beta-unsaturated dihydroxymorpholino derivative is attacked by a nucleophilic residue in the active site.