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Enzyme on ice: Kinetic and EPR spectroscopic characterization of the CoII-substrate radical decay reaction in coenzyme B12-dependent ethanolamine ammonia-lyase
| Content Provider | Semantic Scholar |
|---|---|
| Author | Zhu, Chen |
| Copyright Year | 2010 |
| Abstract | Enzyme on Ice: Kinetic and EPR Spectroscopic Characterization of the Co-Substrate Radical Decay Reaction in Coenzyme B12 By Chen Zhu -Dependent Ethanolamine Ammonia-Lyase The transient decay reaction kinetics of 1,1,2,2-H4and 1,1,2,2-H4-aminoethanolgenerated Co-substrate radical pair catalytic intermediate in ethanolamine ammonialyase (EAL) have been measured by using time-resolved, X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy in frozen aqueous solution from 190 to 223 K. The decay is biexponential at temperature T<214 K (H) or <210 K (H), with fast and slow phase first-order rate constants kobs,f and kobs,s, respectively. The decay becomes monoexponential at temperature T≥214 K, with rate constant kobs,m. The kobs,f and kobs,m values adhere to the same linear relation on a lnk versus T (Arrhenius) plot, and therefore represent the same mechanism, which is proposed to be the native forward reaction of the substrate radical through the radical rearrangement step. The H/H isotope effect (IE) on kobs,f of 1.4±0.1 at 190 ≤T≤207 K is assigned to an α-secondary hydrogen kinetic IE on the rearrangement step. The kobs,s values obey a different Arrhenius relation, and display an inverse kinetic IE (0.8±0.1). The slow decay phase is proposed to be associated with the forward reaction, but with a different rate determining step. The H/H IE on kobs,m increases continuously at T>210 K, to 2.1±0.1 at 223 K. A three-state (substrate radical, product radical, diamagnetic products), two-step [rearrangement, and subsequent hydrogen atom transfer, (HT)] model is used to generate a consistent fit to the temperature dependence of the kobs,f, kobs,m values and IEs at low temperature with kcat values and IEs at 277 K (IE=5.5) and 293 K (IE=7.8). The model shows that the four decade-old paradox of H/H and H/H IEs in EAL, and the temperature dependent IE, are caused by a significant negative activation entropy for the HT step, relative to rearrangement. The bifurcation of the decay kinetics at 207 |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | https://etd.library.emory.edu/downloads/sj1392460?locale=en |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
