Resolution and characterization of contributions of select protein and coupled solvent configurational fluctuations to radical rearrangement catalysis in coenzyme B12-dependent ethanolamine ammonia-lyase.
Methods Enzymol
; 669: 229-259, 2022.
Article
en En
| MEDLINE
| ID: mdl-35644173
ABSTRACT
Coenzyme B12 (adenosylcobalamin) -dependent ethanolamine ammonia-lyase (EAL) is the signature enzyme in ethanolamine utilization metabolism associated with microbiome homeostasis and disease conditions in the human gut. The enzyme conducts a complex choreography of bond-making/bond-breaking steps that rearrange substrate to products through a radical mechanism, with themes common to other coenzyme B12-dependent and radical enzymes. The methods presented are targeted to test the hypothesis that particular, select protein and coupled solvent configurational fluctuations contribute to enzyme function. The general approach is to correlate enzyme function with an introduced perturbation that alters the properties (for example, degree of concertedness, or collectiveness) of protein and coupled solvent dynamics. Methods for sample preparation and low-temperature kinetic measurements by using temperature-step reaction initiation and time-resolved, full-spectrum electron paramagnetic resonance spectroscopy are detailed. A framework for interpretation of results obtained in ensemble systems under conditions of statistical equilibrium within the reacting, globally unstable state is presented. The temperature-dependence of the first-order rate constants for decay of the cryotrapped paramagnetic substrate radical state in EAL, through the chemical step of radical rearrangement, displays a piecewise-continuous Arrhenius dependence from 203 to 295K, punctuated by a kinetic bifurcation over 219-220K. The results reveal the obligatory contribution of a class of select collective protein and coupled solvent fluctuations to the interconversion of two resolved, sequential configurational substates, on the decay time scale. The select class of collective fluctuations also contributes to the chemical step. The methods and analysis are generally applicable to other coenzyme B12-dependent and related radical enzymes.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Etanolamina Amoníaco-Liasa
Límite:
Humans
Idioma:
En
Revista:
Methods Enzymol
Año:
2022
Tipo del documento:
Article
País de afiliación:
Estados Unidos