Formation of the cystine between cysteine 225 and cysteine 462 from ribonucleoside diphosphate reductase is kinetically competent.

Abstract

Participation of the formation of the cystine between cysteine 225 and cysteine 462 in the R1 protein to the enzymatic mechanism of aerobic ribonucleoside diphosphate reductase from Escherichia coli has been examined by use of rapid quenching and site-directed immunochemistry. Prereduced ribonucleotide reductase in the presence of ATP was mixed with CDP in a quench flow apparatus. The reaction was terminated with a solution of acetic acid and N-ethylmaleimide. The protein was precipitated and digested with chymotrypsin and the proteinase from Staphylococcus aureus strain V8 in the presence of N-ethylmaleimide to yield the peptide SS[S-(N-ethylsuccinimid-2-yl)cysteinyl]VLIE containing cysteine 225 and the mixed disulfide between the peptide SSCVLIE and the peptide IALCTL containing cysteine 462. These two peptides were retrieved together from the digest by immunoadsorption. The affinity-purified peptides were modified at their amino termini with the fluorescent reagent 6-aminoquinolyl-N-hydroxysuccimidyl carbamate and submitted to high-pressure liquid chromatography. The areas of the respective peaks of fluorescence corresponding to the S-(N-ethylsuccimidyl) peptide, and the mixed disulfide were used to determine the percentage of the cystine that had formed during each interval. The rate constant for the formation of the cystine following the association of free, fully reduced ribonucleotide reductase with the reactant CDP was 8 s(-)(1). Because only 50% of the active sites participated in this pre-steady-state reaction, the maximum steady-state rate consistent with the involvement of this cystine in the enzymatic reaction would be 4 s(-1). Since the turnover number of the enzyme under the same conditions in a steady state assay was only 1 s(-)(1), the formation of the cystine between these two cysteines is kinetically competent.