Electron spin-echo studies of the composition of the paramagnetic intermediate formed during the deamination of propanolamine by ethanolamine ammonia-lyase, and AdoCbl-dependent enzyme.
“…These studies all reach the same basic conclusion: that homolysis of AdoCbl and hydrogen abstraction from the substrate (or protein in the case of ribonucleotide reductase) are either strongly kinetically coupled or concerted events, requiring that adenosyl radical is never formed at significant concentrations during the catalytic cycle. This finding is in accord with EPR experiments on these and other AdoCbl-dependent enzymes which have failed to detect adenosyl radical ( − ). 1 …”
Glutamate mutase catalyzes the reversible isomerization of L-glutamate to L-threo-3-methylaspartate. Rapid quench experiments have been performed to measure apparent rate constants for several chemical steps in the reaction. The formation of substrate radicals when the enzyme was reacted with either glutamate or methylaspartate was examined by measuring the rate at which 5'-deoxyadenosine was formed, and shown to be sufficiently fast for this step to be kinetically competent. Furthermore, the apparent rate constant for 5'-deoxyadenosine formation was very similar to that measured previously for cleavage of the cobalt-carbon bond of adenosylcobalamin by the enzyme, providing further support for a mechanism in which homolysis of the coenzyme is coupled to hydrogen abstraction from the substrate. The pre-steady-state rates of methylaspartate and glutamate formation were also investigated. No burst phase was observed with either substrate, indicating that product release does not limit the rate of catalysis in either direction. For the conversion of glutamate to methylaspartate, a single chemical step appeared to dominate the overall rate, whereas in the reverse direction a lag phase was observed, suggesting the accumulation of an intermediate, tentatively ascribed to glycyl radical and acrylate. The rates of formation and decay of this intermediate were also sufficiently rapid for it to be kinetically competent. When combined with information from previous mechanistic studies, these results allow a qualitative free energy profile to constructed for the reaction catalyzed by glutamate mutase.
“…These studies all reach the same basic conclusion: that homolysis of AdoCbl and hydrogen abstraction from the substrate (or protein in the case of ribonucleotide reductase) are either strongly kinetically coupled or concerted events, requiring that adenosyl radical is never formed at significant concentrations during the catalytic cycle. This finding is in accord with EPR experiments on these and other AdoCbl-dependent enzymes which have failed to detect adenosyl radical ( − ). 1 …”
Glutamate mutase catalyzes the reversible isomerization of L-glutamate to L-threo-3-methylaspartate. Rapid quench experiments have been performed to measure apparent rate constants for several chemical steps in the reaction. The formation of substrate radicals when the enzyme was reacted with either glutamate or methylaspartate was examined by measuring the rate at which 5'-deoxyadenosine was formed, and shown to be sufficiently fast for this step to be kinetically competent. Furthermore, the apparent rate constant for 5'-deoxyadenosine formation was very similar to that measured previously for cleavage of the cobalt-carbon bond of adenosylcobalamin by the enzyme, providing further support for a mechanism in which homolysis of the coenzyme is coupled to hydrogen abstraction from the substrate. The pre-steady-state rates of methylaspartate and glutamate formation were also investigated. No burst phase was observed with either substrate, indicating that product release does not limit the rate of catalysis in either direction. For the conversion of glutamate to methylaspartate, a single chemical step appeared to dominate the overall rate, whereas in the reverse direction a lag phase was observed, suggesting the accumulation of an intermediate, tentatively ascribed to glycyl radical and acrylate. The rates of formation and decay of this intermediate were also sufficiently rapid for it to be kinetically competent. When combined with information from previous mechanistic studies, these results allow a qualitative free energy profile to constructed for the reaction catalyzed by glutamate mutase.
“…Features at 0.8, 1.8, and 2.8 MHz were previously reported for a single τ and magnetic field value in an X-band ESEEM study of Clostridium sp. ethanolamine deaminase . Figure shows that the feature at 4.0 MHz in Figure increases in frequency to 4.3 MHz when the magnetic field strength is increased.…”
Interactions of the cryotrapped 2-aminopropanol-1-yl substrate radical and aminoethanol-derived
product radical catalytic intermediates with the active site of vitamin B12 coenzyme-dependent ethanolamine
deaminase from Salmonella
typhimurium in disordered frozen aqeuous solution have been characterized by
using X-band electron spin−echo electron paramagnetic resonance (ESE-EPR) and electron spin−echo envelope
modulation (ESEEM) spectroscopies performed at 6 K. ESE-EPR spectra show that the doublet splitting of
the radical line shape by electron−electron exchange and dipolar interactions with CoII in cob(II)alamin is
stronger for the substrate radical (11.1 mT) than for the product radical (7.1 mT). The aminoethanol-derived
product radical unpaired spin density at C2 is therefore positioned closer to CoII than the 2-aminopropanol-1-yl substrate radical unpaired spin density at C1. Multifrequency three-pulse ESEEM spectra obtained at g =
2.00 for each radical display the same τ- and magnetic field-dependent five-line pattern of narrow features.
This indicates that the substrate and product radicals are comparably coupled to the same 14N nucleus. ESEEM
simulations give the following best-fit principal hyperfine tensor and nuclear quadrupole coupling parameters:
A = [−1.1, −0.8, −0.8] MHz; e
2
qQ/h = 3.05 MHz; η = 0.51. The quadrupole parameters are consistent with
a secondary amide nitrogen in a polypeptide bond or with the N10 amino nitrogen in the adenine ring of
5‘-deoxyadenosine. The equivalent 14N coupling for each radical indicates that either the nitrogen nucleus lies
along the C1−C2 bond bisector or the interaction responsible for the coupling moves to remain in register with
the center of unpaired spin density as it shifts 1.5 Å from C1 to C2 in the substrate-to-product radical
transformation. The longer separation distance from CoII of the product versus substrate radical indicates that
the principle of radical pair stabilization by separation operates continuously through the rearrangement reaction.
“…In B12; (32) The identification of the 2-aminopropanol-generated radical as the unrearranged substrate radical 21 was later questioned because electron spinecho envelope modulation (ESEEM) spectra obtained from the radical generated from natural abundance 14 N-aminoethanol and 15 N-labeled aminoethanol were interpreted to be indistinguishable. 33 However, owing to the sensitive dependence of ESEEM amplitudes on the relative contributions of dipolar and isotropic hyperfine coupling contributions, 34 the absence of features from the substrate nitrogen in the ESEEM spectra does not constitute sufficient evidence that the substrate nitrogen is absent from the radical. The ESEEM results 33 Sample Preparation.…”
Section: Methodsmentioning
confidence: 99%
“…33 However, owing to the sensitive dependence of ESEEM amplitudes on the relative contributions of dipolar and isotropic hyperfine coupling contributions, 34 the absence of features from the substrate nitrogen in the ESEEM spectra does not constitute sufficient evidence that the substrate nitrogen is absent from the radical. The ESEEM results 33 Sample Preparation. Adenosylcobalamin (Sigma Chemical Co.), unlabeled aminoethanol (Aldrich Chemical Co.), and isotopicallylabeled aminoethanol (Cambridge Isotope Laboratories, Inc.) were purchased from commercial sources.…”
The assignment of the aminoethanol-derived Co II-radical pair state in ethanolamine deaminase (ethanolamine ammonia-lyase) has been changed to the "Co II-substrate radical pair" from the "Co II-product radical pair", as originally reported. The basis of the reassignment and a supporting nuclear magnetic resonance (NMR) spectrum are presented in Supporting Information. Page 3604 and Table 2. The units for the kinetic parameters of MccB were incorrectly labeled. The units for k cat should read h-1 (not s-1), and the units for k cat /K m should read h-1 µM-1. Errors have also been discovered in the reported kinetic parameters for MccB in Table 2. The k cat for succinimide kinetics is 296 (48 h-1 , and k cat /K m) 2.34 h-1 µM-1 , making the conversion of succinimide 4 to product 3 ∼40-fold faster than the conversion of 2 to 3.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
