ABSTRACT
Ribonucleotide reductase (RNR), which supplies the building blocks for DNA biosynthesis and its repair, has been linked to human diseases and is emerging as a therapeutic target. Here, we present a mechanistic investigation of triapine (3AP), a clinically relevant small molecule that inhibits the tyrosyl radical within the RNR ß2 subunit. Solvent kinetic isotope effects reveal that proton transfer is not rate-limiting for inhibition of Y122· of E. coli RNR ß2 by the pertinent 3AP-Fe(II) adduct. Vibrational spectroscopy further demonstrates that unlike inhibition of the ß2 tyrosyl radical by hydroxyurea, a carboxylate containing proton wire is not at play. Binding measurements reveal a low nanomolar affinity (Kd â¼ 6 nM) of 3AP-Fe(II) for ß2. Taken together, these data should prompt further development of RNR inactivators based on the triapine scaffold for therapeutic applications.
Subject(s)
Enzyme Inhibitors/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Ferrous Compounds/chemistry , Pyridines/chemistry , Ribonucleotide Reductases/metabolism , Thiosemicarbazones/chemistry , Enzyme Inhibitors/metabolism , Escherichia coli Proteins/antagonists & inhibitors , Free Radicals/chemistry , Free Radicals/metabolism , Hydroxyurea/chemistry , Protein Binding , Protein Subunits/antagonists & inhibitors , Protein Subunits/chemistry , Protein Subunits/metabolism , Ribonucleotide Reductases/antagonists & inhibitors , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform InfraredABSTRACT
Ribonucleotide reductase (RNR) catalyzes the production of deoxyribonucleotides in all cells. In E. coli class Ia RNR, a transient α2ß2 complex forms when a ribonucleotide substrate, such as CDP, binds to the α2 subunit. A tyrosyl radical (Y122Oâ¢)-diferric cofactor in ß2 initiates substrate reduction in α2 via a long-distance, proton-coupled electron transfer (PCET) process. Here, we use reaction-induced FT-IR spectroscopy to describe the α2ß2 structural landscapes, which are associated with dATP and hydroxyurea (HU) inhibition. Spectra were acquired after mixing E. coli α2 and ß2 with a substrate, CDP, and the allosteric effector, ATP. Isotopic chimeras, (13)Cα2ß2 and α2(13)Cß2, were used to define subunit-specific structural changes. Mixing of α2 and ß2 under turnover conditions yielded amide I (CâO) and II (CN/NH) bands, derived from each subunit. The addition of the inhibitor, dATP, resulted in a decreased contribution from amide I bands, attributable to ß strands and disordered structures. Significantly, HU-mediated reduction of Y122O⢠was associated with structural changes in α2, as well as ß2. To define the spectral contributions of Y122Oâ¢/Y122OH in the quaternary complex, (2)H4 labeling of ß2 tyrosines and HU editing were performed. The bands of Y122Oâ¢, Y122OH, and D84, a unidentate ligand to the diferric cluster, previously identified in isolated ß2, were observed in the α2ß2 complex. These spectra also provide evidence for a conformational rearrangement at an additional ß2 tyrosine(s), Yx, in the α2ß2/CDP/ATP complex. This study illustrates the utility of reaction-induced FT-IR spectroscopy in the study of complex enzymes.
