Characterization of azido-NAD+ to assess its potential as a two-dimensional infrared probe of enzyme dynamics

Dutta, Samrat; Cook, Richard J.; Houtman, Jon C. D.; Kohen, Amnon; Cheatum, Christopher M.

doi:10.1016/j.ab.2010.08.008

Cited by 20 publications

(35 citation statements)

References 44 publications

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“…The assignment of Fermi resonance is in agreement with a previous literature study, 28 although 2D IR studies of N 3 CNdU are needed for confirmation. 27, 28, 48–53 …”

Section: Resultsmentioning

confidence: 99%

A direct comparison of azide and nitrile vibrational probes

Gai

Coutifaris

Brewer

et al. 2011

Phys. Chem. Chem. Phys.

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The synthesis of 2′-azido-5-cyano-2′-deoxyuridine, N3CNdU (1), from trityl-protected 2′-amino-2′-deoxyuridine was accomplished in four steps with a 12.5% overall yield. The IR absorption positions and profiles of the azide and nitrile group of N3CNdU were investigated in 14 different solvents and water/DMSO solvent mixtures. The azide probe was superior to the nitrile probe in terms of its extinction coefficient, which is 2–4 times larger. However, the nitrile IR absorbance profile is generally less complicated by accidental Fermi resonance. The IR frequencies of both probes undergo a substantial red shift upon going from water to aprotic solvents such as THF or DMSO. DFT calculations supported the hypothesis that the molecular origin of the higher observed frequency in water is primarily due to hydrogen bonds between the probes and water molecules.

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“…The assignment of Fermi resonance is in agreement with a previous literature study, 28 although 2D IR studies of N 3 CNdU are needed for confirmation. 27, 28, 48–53 …”

Section: Resultsmentioning

confidence: 99%

A direct comparison of azide and nitrile vibrational probes

Gai

Coutifaris

Brewer

et al. 2011

Phys. Chem. Chem. Phys.

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“…In addition, because azide will not mimic the TRS in other systems and only binds a limited number of enzymes, other probes would also offer access to the dynamics in other systems. To this end, we have prepared and characterized azido-labeled analogs of NAD + , azido-NAD + , 103,104 in which the amide group on the nicotinamide ring has been replaced with an azide, and picolyl azide adenine dinucleotide (PAAD + ) in which the amide group is replaced by an azide that is separated from the pyridinium ring by a methylene spacer. 105 Azido-NAD + is attractive because it is a smaller perturbation than is PAAD + , but PAAD + has a significantly stronger transition moment.…”

Section: Fdh – Combining the Kinetic And Spectroscopic Methodsmentioning

confidence: 99%

Relationship of Femtosecond–Picosecond Dynamics to Enzyme-Catalyzed H-Transfer

Cheatum

Kohen

2013

Dynamics in Enzyme Catalysis

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At physiological temperatures, enzymes exhibit a broad spectrum of conformations, which interchange via thermally activated dynamics. These conformations are sampled differently in different complexes of the protein and its ligands, and the dynamics of exchange between these conformers depends on the mass of the group that is moving and the length scale of the motion, as well as restrictions imposed by the globular fold of the enzymatic complex. Many of these motions have been examined and their role in the enzyme function illuminated, yet most experimental tools applied so far have identified dynamics at time scales of seconds to nanoseconds, which are much slower than the time scale for H-transfer between two heavy atoms. This chemical conversion and other processes involving cleavage of covalent bonds occur on picosecond to femtosecond time scales, where slower processes mask both the kinetics and dynamics. Here we present a combination of kinetic and spectroscopic methods that may enable closer examination of the relationship between enzymatic C-H→C transfer and the dynamics of the active site environment at the chemically relevant time scale. These methods include kinetic isotope effects and their temperature dependence, which are used to study the kinetic nature of the H-transfer, and 2D IR spectroscopy, which is used to study the dynamics of transition-state- and ground-state-analog complexes. The combination of these tools is likely to provide a new approach to examine the protein dynamics that directly influence the chemical conversion catalyzed by enzymes.

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“…We have recently shown that an azido-derivatized analog of NAD + , azido-NAD + , has the potential to be a general probe to investigate the active site dynamics of NAD-dependent enzymes. 40 This analog exhibits binding and kinetic properties that are similar to native NAD + for a number of enzymes, suggesting that the analog binds to the enzyme with minimal perturbations to the active site structure. For azido-NAD + we have also studied its spectroscopic characteristics.…”

Section: Introductionmentioning

confidence: 93%

3-Picolyl Azide Adenine Dinucleotide as a Probe of Femtosecond to Picosecond Enzyme Dynamics

Dutta

Rock

et al. 2011

J. Phys. Chem. B

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Functionally relevant femtosecond to picosecond dynamics in enzyme active sites can be difficult to measure because of a lack of spectroscopic probes that can be located in the active site without altering the behavior of the enzyme. We have developed a new NAD+ analog 3-Picolyl Azide Adenine Dinucleotide (PAAD+), which has the potential to be a general spectroscopic probe for NAD-dependent enzymes. This analog is stable and binds in the active site of a typical NAD-dependent enzyme formate dehydrogenase (FDH) with similar characteristics to natural NAD+. It has an isolated infrared transition with high molar absorptivity that makes it suitable for observing enzyme dynamics using 2D IR spectroscopy. 2D IR experiments show that in aqueous solution, the analog undergoes complete spectral diffusion within hundreds of femtoseconds consistent with the water hydrogen bonding dynamics that would be expected. When bound to FDH in a binary complex, it shows picosecond fluctuations and a large static offset, consistent with previous studies of the binary complexes of this enzyme. These results show that PAAD+ is an excellent probe of local dynamics and that it should be a general tool for probing the dynamics of a wide range of NAD-dependent enzymes.

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Characterization of azido-NAD+ to assess its potential as a two-dimensional infrared probe of enzyme dynamics

Cited by 20 publications

References 44 publications

A direct comparison of azide and nitrile vibrational probes

A direct comparison of azide and nitrile vibrational probes

Relationship of Femtosecond–Picosecond Dynamics to Enzyme-Catalyzed H-Transfer

3-Picolyl Azide Adenine Dinucleotide as a Probe of Femtosecond to Picosecond Enzyme Dynamics

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