| |
[摘要]：Echerichia coli ribonucleotide reductase (RNR), an alpha 2 beta 2 complex, catalyzes the conversion of nucleoside 5'-diphosphate Substrates (S) to 2'-deoxynucleoside 5'-diphosphates. alpha 2 houses the active site for nucleotide reduction and the binding sites For allosteric effectors (E). beta 2 contains the essential diferric tyrosyl radical (Y-122(center dot)) cofactor which, in the presence of S and E, oxidizes C-439 in alpha to a thiyl radical, C-439(center dot) to initiate nucleotide reduction. This oxidation occurs over 35 angstrom and is proposed to involve a specific pathway: Y-122(center dot) -> W-48 -> Y-356 in beta(2) to Y-731 -> Y-730 -> C-439 in alpha 2. 3-Aminotyrosine (NH2Y) has been site-specifically incorporated at residues 730 and 731, and formation of the aminotyrosyl radical (NH2Y center dot) has been examined by stopped-flow (SF) UV-vis and EPR spectroscopies. To examine the pathway dependence of radical propagation, the double Mutant complexes Y356F-beta 2:Y731NH2Y-alpha 2, Y356F-beta(2):Y730NH2Y-alpha 2, and wt-beta 2:Y731F/Y730NH2Y-alpha 2 , in which the nonoxidizable F acts its a pathway block, were studied by SF and EPR spectroscopies. In all cases, no NH2Y center dot was detected. To study off-pathway oxidation, Y-413, located 5 angstrom from Y-730 and Y-731 but not implicated in long-range oxidation, was examined. Evidence for NH2Y413 center dot was Sought ill three complexes: wt-beta 2:Y413NH2Y-alpha 2 (a), wt-beta 2:Y731F/Y413NH2Y-alpha(2) (b), and Y356F-beta(2): Y413NH2Y-alpha(2) (c). With (a), NH2Y center dot was formed with a rate constant that was 25-30% and ail amplitude that was 25% of that observed for its formation at residues 731 and 730. With (b), the rate constant for NH2Y center dot formation was 0.2-0.3% of that observed at 731 and 730, and with (c), no NH2Y center dot was observed. These studies suggest the evolution of ail optimized pathway of conserved Ys in the oxidation of C-439. |
|
