期刊
JOURNAL OF CHEMICAL PHYSICS
卷 154, 期 17, 页码 -出版社
AMER INST PHYSICS
DOI: 10.1063/5.0040341
关键词
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资金
- National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH) [R01 DK054514]
- National Institute of General Medical Sciences (NIGMS) of the NIH [R01 GM142113]
- National Center for Research Resources of the NIH [RR17767]
- Emory University
EPR spectroscopy was used to study the substrate radical rearrangement reaction of B-12-dependent ethanolamine ammonia-lyase at physiological and cryogenic temperatures. The study demonstrated the formation of aqueous-cosolvent mesodomain and protein-associated domain around EAL in frozen solution. Changes in the solvent domain affected the EPR amplitude of cob(II)alamin and the substrate radical, influencing enzyme reactivity at cryogenic temperatures.
Electron paramagnetic resonance (EPR) spectroscopy is used to address the remarkable persistence of the native Arrhenius dependence of the 2-aminopropanol substrate radical rearrangement reaction in B-12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium from physiological to cryogenic (220 K) temperatures. Two-component TEMPOL spin probe mobility in the presence of 10 mM (0.08% v/v) 2-aminopropanol over 200-265 K demonstrates characteristic concentric aqueous-cosolvent mesodomain and protein-associated domain (PAD, hydration layer) solvent phases around EAL in the frozen solution. The mesodomain formed by the relatively small amount of 2-aminopropanol is highly confined, as shown by an elevated temperature for the order-disorder transition (ODT) in the PAD (230-235 K) and large activation energy for TEMPOL rotation. Addition of 2% v/v dimethylsulfoxide expands the mesodomain, partially relieves PAD confinement, and leads to an ODT at 205-210 K. The ODT is also manifested as a deviation of the temperature-dependence of the EPR amplitude of cob(II)alamin and the substrate radical, bound in the enzyme active site, from Curie law behavior. This is attributed to an increase in sample dielectric permittivity above the ODT at the microwave frequency of 9.5 GHz. The relatively high frequency dielectric response indicates an origin in coupled protein surface group-water fluctuations of the Johari-Goldstein beta type that span spatial scales of similar to 0.1-10 angstrom on temporal scales of 10(-10)-10(-7) s. The orthogonal EPR spin probe rotational mobility and solvent dielectric measurements characterize features of EAL protein-solvent dynamical coupling and reveal that excess substrate acts as a fluidizing cryosolvent to enable native enzyme reactivity at cryogenic temperatures.
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