Mechanistic investigations of class I ribonucleotide reductases and related oxygen-utilizing metalloenzymes.

摘要

Enzymes, nature's preferred catalysts, often effect remarkable transformations of their substrates, and the mechanistic elucidation of these catalysts has significantly advanced our understanding of biological processes. Our work is focuses on the detailed characterization of enzymes operating in an aerobic environment that utilize one or more transition metals as cofactors to facilitate transformation of their substrates. The approach of choice to facilitate the mechanistic elucidation of these enzymatic reactions involves the capture of transient species (reactive intermediates) that occur along each reaction sequence, and the kinetic and spectroscopic characterization of each captured species. A major focus of this thesis involves mechanistic investigations of a classic O2-utilizing metalloenzyme, ribonucleotide reductase (RNR).;By catalyzing the conversion of ribonucleotides to deoxyribonucleotides [ND(T)Ps], RNRs provide all organisms with the required precursors for the de novo synthesis and repair of DNA. RNRs accomplish this chemically challenging feat with great fidelity by harnessing free-radical chemistry. To date, all RNRs characterized utilize an unstable cysteine thiyl radical (C·), formed in close proximity to the bound ND(T)P to initiate ribonucleotide reduction. Class I RNRs, which all of our studies are focused on and encompass the enzymes from all mammals, aerobically-growing Escherichia coli (Ec), and the human pathogen Chlamydia trachomatis (Ct), are comprised of two non-identical protein subunits, termed alpha and beta. The alpha subunit contains the oxidizable C residue and the site of nucleotide reduction, whereas the beta subunit assembles a metallocofactor cofactor, ∼ 35 A away from the site of catalysis in alpha. To reduce ribonucleotides, all RNRs must transfer an oxidizing equivalent or 'hole', stored at the metallocofactor in beta, to alpha and generate the C· in a reversible process. The identity of metallocofactor is key distinguishing factor between the subclasses of class I RNRs, with the Ia and Ib enzymes utilizing diiron- and dimanganese-tyrosyl radical cofactors, and Ic employing a MnIV/FeIII cofactor.;The second part of this thesis focuses on the development of a method with utility that extends beyond our studies on Ct RNR. The capture (by rapid-mixing kinetic techniques) and characterization (by spectroscopy) of fleeting, reactive intermediates is the most-favored approach to mechanistic dissection of metalloenzymes. For metalloenzymes that also utilize molecular oxygen, O2, this approach is often hampered by the gas's modest aqueous solubility, which, at < 2 mM (at 1 atmosphere) limits both the effective rate constants for formation of reactive intermediates and the concentrations to which the intermediates can accumulate. Our work presented in Appendix C sought to overcome the challenge imposed by the poor solubility of O2 by using the enzyme chlorite dismutase (Cld), for the rapid, in situ generation of O2 at concentrations far exceeding 2 mM. Cld, a heme enzyme, efficiently converts chlorite (ClO 2-) to O2 and chloride ion (Cl-). The method a) permits accumulation of O2-derived complexes at concentrations well above 2 mM, b) allows greater precision in determining the O2- dependent kinetics of enzymes that bind or react with O2, and c) permits substantial increase in the yield of intermediates that form in a reversible, disfavored equilibrium with O2. This means of in situ O2 generation permits a > 5 mM "pulse" of O2 to be generated in < 1 ms at the easily accessible [Cld] of 50 muM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O2- addition steps. (Abstract shortened by UMI.).