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Dynamic, electrostatic model for the generation and control of high-energy radical intermediates by a coenzyme B₁₂-dependent enzyme.

Chen ZG, Ziętek MA, Russell HJ, Tait S, Hay S, Jones AR, Scrutton NS - Chembiochem (2013)

View Article: PubMed Central - PubMed

Affiliation: College of Food and Science Technology, Nanjing Agricultural University, 1 Weigang Road, Nanjing 210095 (P.R. China).

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In the case of coenzyme B12-dependent enzymes, this energy is “lent” in the form of radical intermediates, which are formed upon substrate binding by Co–C bond homolysis in the cofactor... Herein, we present data that suggest that a single point mutation can have a significant impact on Co–C bond homolysis and the control of the radical trajectory and reactivity in coenzyme B12-dependent ethanolamine ammonia lyase (EAL)... The peak of the αβ absorption band for free coenzyme B12 in aqueous buffer is at 525 nm and that for methylcobalamin is at 521 nm... These peaks were found to blue-shift by varying amounts when bound to wild-type (WT) EAL and the E287 variants (Figures 2 and S3 as well as Table S1)... Initially, therefore, these data were acquired under aerobic conditions... There is a substantial decrease in the apparent kcat in both E287D (∼100-fold) and E287Q (∼820-fold) compared to WT... If radical pair recombination is required after each turnover, one might expect either deactivation after a single turnover or a substantial reduction in turnover of E287Q under aerobic conditions... Instead, we only see a slight reduction in kcat upon the introduction of oxygen to the reaction sample, this suggests a significant proportion of radical pairs remain dissociated until the substrate is exhausted... However, a deuterium KIE of ∼7 has been reported on turnover of 2-aminoethanol by EAL, thus indicating that H transfer from the coenzyme to the product-like radical must also contribute, at least partially, to the overall rate... The effect on these steps warrants further investigation... This is an interesting result because published computational data suggest that the electrostatic contribution to Co–C bond homolysis is dominant in coenzyme B12-dependent mutases... Such generalised local flexibility in EAL therefore provides a dynamic contribution to the electrostatic model of Co–C bond homolysis in B12-dependent enzymes... A nearby arginine, R160 (Figure 1), has previously been shown as playing a critical role in EAL catalysis... Again, the formal (positive) charge of R160 is significant, with R160K retaining activity with a modest drop in kcat/Km (∼180-fold) and R160A resulting in the accumulation of cob(II)alamin but not facilitating turnover... This picture supports the model for Co–C bond homolysis in EAL proposed by Robertson et al.: coordinated protein motions that guide the cleavage of the Co–C bond along the reaction coordinate.

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Peak wavelengths of the αβ absorption band for coenzyme B12 (5′-deoxyadenosylcobalamin, black bars) and methylcobalalmin (grey bars) both free in aqueous buffer and bound to WT EAL and the E287D/Q/A variants. The EAL variants are arranged from left to right in order of increasing hydrophobicity of residue 287 (Asp<Glu<Gln<Ala).[10] The ±2 nm error was based on the breadth of the peaks. See text for discussion.
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fig02: Peak wavelengths of the αβ absorption band for coenzyme B12 (5′-deoxyadenosylcobalamin, black bars) and methylcobalalmin (grey bars) both free in aqueous buffer and bound to WT EAL and the E287D/Q/A variants. The EAL variants are arranged from left to right in order of increasing hydrophobicity of residue 287 (Asp<Glu<Gln<Ala).[10] The ±2 nm error was based on the breadth of the peaks. See text for discussion.

Mentions: The peak of the αβ absorption band for free coenzyme B12 in aqueous buffer is at 525 nm and that for methylcobalamin is at 521 nm. These peaks were found to blue-shift by varying amounts when bound to wild-type (WT) EAL and the E287 variants (Figures 2 and S3 as well as Table S1). The magnitude of the shift is similar within error (∼4 nm) for coenzyme B12 bound to WT, E287D and E287Q. Such a shift suggests a more hydrophobic environment for the B12 chromophore when bound to the protein than in aqueous solution. This hydrophobic effect is enhanced greatly in E287A (a (15±2) nm blue shift). On the other hand, the average blue shift is ∼7 nm for protein-bound methylcobalamin and is comparable within error across the EAL variants. These data suggest a strong influence of E287 on coenzyme B12, through direct contact with the upper axial 5′-deoxyadenosyl that is not facilitated by the smaller upper axial methyl. Moreover, there is greater general variation between the spectra of B12 bound to the EAL variants than between those of protein-bound methylcobalamin (Figure S3 A and C). Contact between E287 and 5′-deoxyadenosyl might be the origin of the ultrafast vibrational coupling observed between photoexcited B12 and EAL.[9a]


Dynamic, electrostatic model for the generation and control of high-energy radical intermediates by a coenzyme B₁₂-dependent enzyme.

Chen ZG, Ziętek MA, Russell HJ, Tait S, Hay S, Jones AR, Scrutton NS - Chembiochem (2013)

Peak wavelengths of the αβ absorption band for coenzyme B12 (5′-deoxyadenosylcobalamin, black bars) and methylcobalalmin (grey bars) both free in aqueous buffer and bound to WT EAL and the E287D/Q/A variants. The EAL variants are arranged from left to right in order of increasing hydrophobicity of residue 287 (Asp<Glu<Gln<Ala).[10] The ±2 nm error was based on the breadth of the peaks. See text for discussion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4155860&req=5

fig02: Peak wavelengths of the αβ absorption band for coenzyme B12 (5′-deoxyadenosylcobalamin, black bars) and methylcobalalmin (grey bars) both free in aqueous buffer and bound to WT EAL and the E287D/Q/A variants. The EAL variants are arranged from left to right in order of increasing hydrophobicity of residue 287 (Asp<Glu<Gln<Ala).[10] The ±2 nm error was based on the breadth of the peaks. See text for discussion.
Mentions: The peak of the αβ absorption band for free coenzyme B12 in aqueous buffer is at 525 nm and that for methylcobalamin is at 521 nm. These peaks were found to blue-shift by varying amounts when bound to wild-type (WT) EAL and the E287 variants (Figures 2 and S3 as well as Table S1). The magnitude of the shift is similar within error (∼4 nm) for coenzyme B12 bound to WT, E287D and E287Q. Such a shift suggests a more hydrophobic environment for the B12 chromophore when bound to the protein than in aqueous solution. This hydrophobic effect is enhanced greatly in E287A (a (15±2) nm blue shift). On the other hand, the average blue shift is ∼7 nm for protein-bound methylcobalamin and is comparable within error across the EAL variants. These data suggest a strong influence of E287 on coenzyme B12, through direct contact with the upper axial 5′-deoxyadenosyl that is not facilitated by the smaller upper axial methyl. Moreover, there is greater general variation between the spectra of B12 bound to the EAL variants than between those of protein-bound methylcobalamin (Figure S3 A and C). Contact between E287 and 5′-deoxyadenosyl might be the origin of the ultrafast vibrational coupling observed between photoexcited B12 and EAL.[9a]

View Article: PubMed Central - PubMed

Affiliation: College of Food and Science Technology, Nanjing Agricultural University, 1 Weigang Road, Nanjing 210095 (P.R. China).

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

In the case of coenzyme B12-dependent enzymes, this energy is “lent” in the form of radical intermediates, which are formed upon substrate binding by Co–C bond homolysis in the cofactor... Herein, we present data that suggest that a single point mutation can have a significant impact on Co–C bond homolysis and the control of the radical trajectory and reactivity in coenzyme B12-dependent ethanolamine ammonia lyase (EAL)... The peak of the αβ absorption band for free coenzyme B12 in aqueous buffer is at 525 nm and that for methylcobalamin is at 521 nm... These peaks were found to blue-shift by varying amounts when bound to wild-type (WT) EAL and the E287 variants (Figures 2 and S3 as well as Table S1)... Initially, therefore, these data were acquired under aerobic conditions... There is a substantial decrease in the apparent kcat in both E287D (∼100-fold) and E287Q (∼820-fold) compared to WT... If radical pair recombination is required after each turnover, one might expect either deactivation after a single turnover or a substantial reduction in turnover of E287Q under aerobic conditions... Instead, we only see a slight reduction in kcat upon the introduction of oxygen to the reaction sample, this suggests a significant proportion of radical pairs remain dissociated until the substrate is exhausted... However, a deuterium KIE of ∼7 has been reported on turnover of 2-aminoethanol by EAL, thus indicating that H transfer from the coenzyme to the product-like radical must also contribute, at least partially, to the overall rate... The effect on these steps warrants further investigation... This is an interesting result because published computational data suggest that the electrostatic contribution to Co–C bond homolysis is dominant in coenzyme B12-dependent mutases... Such generalised local flexibility in EAL therefore provides a dynamic contribution to the electrostatic model of Co–C bond homolysis in B12-dependent enzymes... A nearby arginine, R160 (Figure 1), has previously been shown as playing a critical role in EAL catalysis... Again, the formal (positive) charge of R160 is significant, with R160K retaining activity with a modest drop in kcat/Km (∼180-fold) and R160A resulting in the accumulation of cob(II)alamin but not facilitating turnover... This picture supports the model for Co–C bond homolysis in EAL proposed by Robertson et al.: coordinated protein motions that guide the cleavage of the Co–C bond along the reaction coordinate.

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