A key step in the mechanism of all adenosylcobalamin-dependent enzymes is the abstraction of a hydrogen atom from the substrate by a 5'-deoxyadenosyl radical generated by homolytic fission of the coenzyme cobalt-carbon bond. We have investigated the isotope effects associated with this process for glutamate mutase reacting with deuterated glutamate. The kinetics of deuterium incorporation into 5'-deoxyadenosine (5'-dA) during the reaction were followed by rapid chemical quench, using HPLC and electrospray mass spectrometry to analyze the 5'-dA formed. The kinetics of 5'-dA formation are biphasic, comprising a rapid phase k(app) = 37 +/- 3 s(-)(1) and a slower phase k(app) = 0.9 +/- 0.4 s(-)(1). The mass spectral data clearly show that the faster phase is associated with the formation of monodeuterated 5'-dA whereas the slower phase is associated with the incorporation of a second and then a third deuterium into 5'-dA. This observation implies that a large inverse equilibrium secondary isotope effect is associated with the formation of 5'-dA from adenosylcobalamin. The primary deuterium kinetic isotope effects on V and V/K for the formation of 5'-dA were determined from time-based and competition experiments. (D)V = 2.4 +/-0.4 whereas (D)(V/K) = 10 +/- 0.4, implying that an isotopically insensitive step is partially rate-determining. The additional data provided by these experiments cause us to revise our interpretation of earlier UV-visible stopped-flow kinetic measurements of AdoCbl homolysis obtained with deuterated substrates.

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze a variety of reactions that proceed through organic radical intermediates generated by homolytic fission of coenzyme's unique cobalt-carbon bond. For all the enzymes that have been examined, the homolysis step is kinetically indistinguishable from abstraction of hydrogen from the substrate (or protein), implying that deoxyadenosyl radical is formed only as a fleeting intermediate. To examine how these two steps are coupled together, we have used pre-steady-state, rapid quench techniques to measure the alpha-secondary tritium isotope effect associated with the formation of 5'-deoxyadenosine when the enzyme is reacted with [5'-(3)H]-adenosylcobalamin and L-glutamate. Surprisingly, a large inverse equilibrium isotope effect of 0.72 +/- 0.04 was found for the overall reaction, indicating that the 5'-C-H bonds become significantly stiffer on going from adenosylcobalamin to 5'-deoxyadenosine, even though the 5'-carbon remains formally sp(3) hybridized. The kinetic isotope effect for the formation of 5'-deoxyadenosine was 0.76 +/- 0.02, which suggests a late transition state for the reaction.

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through organic radical intermediates generated by homolytic fission of coenzyme's unique cobalt-carbon bond. These enzymes are part of a larger family of enzymes that catalyze radical chemistry in which a key step is the abstraction of a hydrogen atom from an otherwise inert substrate. To gain insight into the mechanism of hydrogen transfer we previously used presteady state, rapid quench techniques to measure the α-secondary tritium kinetic and equilibrium isotope effects associated with the formation of 5'-deoxyadenosine when glutamate mutase was reacted with [5'-3 H]-adenosylcobalamin and L-glutamate. We showed that both the kinetic and equilibrium isotope effects are large and inverse, 0.76 and 0.72 respectively. We have now repeated these measurements using glutamate deuterated in the position of hydrogen abstraction. The effect of introducing a primary deuterium kinetic isotope effect on the hydrogen transfer step is to reduce the magnitude of the secondary kinetic isotope effect to a value close to unity, 1.05 ± 0.08, whereas the equilibrium isotope effect is unchanged. The significant reduction in the secondary kinetic isotope effect is consistent with motions of the 5'-hydrogen atoms being coupled in the transition state to the motion of the hydrogen undergoing transfer, in a reaction that involves a large degree of quantum tunneling. Keywordsenzyme; coenzyme-B 12 ; protein-radical; isomerization; transition state; secondary isotope effects Glutamate mutase is one of a group of adenosylcobalamin 1 (AdoCbl, coenzyme B 12 ) dependent enzymes that catalyze unusual carbon skeleton isomerisations. These rearrangements formally involve a 1,2 hydrogen atom migration and proceed through a mechanism involving carbon-based free radical intermediates (1-6). The initial steps of these reactions involve homolysis of the reactive cobalt-carbon bond of the coenzyme to form cob (II)alamin and 5'-deoxyadenosyl radical. The adenosyl radical then abstracts the migrating hydrogen from the substrate to form 5-deoxyadenosine and substrate radical (or protein radical in the case of AdoCbl-dependent ribonucleotide reductase). These steps have been studied in some detail for several B 12 enzymes and in each case homolysis and hydrogen abstraction are found to be kinetically coupled (7-10), as evidenced by the appearance of a kinetic isotope

Single-molecule super-resolution microscopy is an emerging technique for nanometer-scale fluorescence imaging, but in vitro single-molecule imaging protocols typically require a constant supply of reagents, and such transport is restricted in constrained geometries. In this paper, we develop Single-Molecule Micelle-Assisted Blink (MAB) microcopy to enable sub-diffraction-limit imaging of nanochannels with better than 40-nm accuracy. The method, based on micelles and thiol-related photoswitching, is used to measure nanochannels formed in polydimethyl-siloxane (PDMS) through tensile cracking. These conduits are reversibly size-adjustable from a few nanometers up to a micron, and enable filtering of small particles and linearization of DNA. Unfortunately, conventional techniques cannot be used to measure widths, characterize heterogeneities or discover porosity in situ. We overcome the access barriers by using sodium dodecyl sulfate (SDS), an ionic surfactant, to facilitate delivery of Cy5 dye and beta-mercaptoethanol reducing agent in the confined geometry. These SDS micelles and admicelles have the further benefit of slowing diffusion of Cy5 to improve localization accuracy. We use MAB microscopy to measure nanochannel widths, to reveal heterogeneity along channel lengths and between different channels in the same device, and to probe biologically relevant information about the nano-environment, such as solvent accessibility.