title: Studies on ribonucleoside-diphosphate reductase from Escherichia coli. The product dCDP is a competitive inhibitor and functions as a spectroscopic probe for the substrate binding site; demonstration by enzyme kinetics and 1H NMR.
abstract: Ribonucleoside-diphosphate reductase (EC 1.17.4.1) from Escherichia coli consists of two protein subunits, R1 of 171.5 kDa and R2 of 86.8 kDa, and catalyzes the reduction of all four common ribonucleoside diphosphates. In a search for ligands that bind weakly to the enzyme active site and may be in fast exchange suitable for NMR studies, we have found that the product dCDP is a competitive inhibitor. Kinetics with CDP as substrate shows Km = 4.8 x 10(-5) M and dCDP inhibits with Ki = 1.6 x 10(-4) M. With an assumed diffusion limited binding rate approximately less than 10(9) M-1s-1, the dissociation rate of dCDP would be approximately less than 10(5) s-1. In 1H-NMR experiments studying linewidths, i.e. spin-spin relaxation, dCDP is indeed demonstrated to be in fast exchange. Enzyme subunit R1 causes a line broadening of dCDP resonances. Unexpectedly less broadening was observed when subunit R2 combined with R1. No paramagnetic interaction from the tyrosyl radical of R2 could be detected. It is concluded that dCDP is a promising NMR probe for studies of active-site properties of the enzyme.
authors.affiliation: Department of Biophysics, Stockholm University, Arrhenius Laboratory, Sweden.
authors.forename: B
authors.initials: B
authors.lastname: Shen
authors.forename: P
authors.initials: P
authors.lastname: Allard
authors.forename: S
authors.initials: S
authors.lastname: Kuprin
authors.forename: A
authors.initials: A
authors.lastname: Ehrenberg
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D003845:Deoxycytosine Nucleotides / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D012262:Ribonucleoside Diphosphate Reductase / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D003845:Deoxycytosine Nucleotides; C024107:2'-deoxycytidine 5'-triphosphate; D012262:Ribonucleoside Diphosphate Reductase