Pre-steady-state and steady-state kinetic analysis of E. coli class I ribonucleotide reductase.
E. coli ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates (NDPs) to dNDPs and is composed of two homodimeric subunits: R1 and R2. R1 binds NDPs and contains binding sites for allosteric effectors that control substrate specificity and turnover rate. R2 contains a diiron-tyrosyl radical (Y(*)) cofactor that initiates nucleotide reduction. Pre-steady-state experiments with wild type R1 or C754S/C759S-R1 and R2 were carried out to determine which step(s) are rate-limiting and whether both active sites of R1 can catalyze nucleotide reduction. Rapid chemical quench experiments monitoring dCDP formation gave k(obs) of 9 +/- 4 s(-1) with an amplitude of 1.7 +/- 0.4 equiv. This amplitude, generated in experiments with pre-reduced R1 (3 or 15 microM) in the absence of reductant, indicates that both monomers of R1 are active. Stopped-flow UV-vis spectroscopy monitoring the concentration of the Y(*) failed to reveal any changes from 2 ms to seconds under similar conditions. These pre-steady-state experiments, in conjunction with the steady-state turnover numbers for dCDP formation of 2-14 s(-1) at RNR concentrations of 0.05-0.4 microM (typical assay conditions), reveal that the rate-determining step is a physical step prior to rapid nucleotide reduction and rapid tyrosine reoxidation to Y(*). Steady-state experiments conducted at RNR concentrations of 3 and 15 microM, typical of pre-steady-state conditions, suggest that, in addition to the slow conformational change(s) prior to chemistry, re-reduction of the active site disulfide to dithiol or a conformational change accompanying this process can also be rate-limiting.
Ge
Jie
J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Yu
Guixue
G
Ator
Mark A
MA
Stubbe
JoAnne
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D003845:Deoxycytosine Nucleotides / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D011487:Protein Conformation; D021122:Protein Subunits; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry; D013879:Thioredoxins / Q000737:chemistry; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism
D003845:Deoxycytosine Nucleotides; D005609:Free Radicals; D021122:Protein Subunits; D014443:Tyrosine; D013879:Thioredoxins; D009249:NADP; D000255:Adenosine Triphosphate; D012264:Ribonucleotide Reductases; D013880:Thioredoxin-Disulfide Reductase