[NMR paper] Polarization of cinnamoyl-CoA substrates bound to enoyl-CoA hydratase: correlation of

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Polarization of cinnamoyl-CoA substrates bound to enoyl-CoA hydratase: correlation of (13)C NMR with quantum mechanical calculations and calculation of electronic strain energy.

Related Articles Polarization of cinnamoyl-CoA substrates bound to enoyl-CoA hydratase: correlation of (13)C NMR with quantum mechanical calculations and calculation of electronic strain energy.

Biochemistry. 2002 Feb 26;41(8):2630-40

Authors: D'Ordine RL, Pawlak J, Bahnson BJ, Anderson VE

When alpha,beta-unsaturated substrates bind to the active site of enoyl-CoA hydratase, large spectral changes can be observed [D'Ordine, R. L., et al. (1994) Biochemistry 33, 12635-12643]. The differences in the isotropic magnetic shieldings of the free and active site-bound forms of the carbonyl, alpha-, and beta-carbons of the substrates, hexadienoyl-CoA, cinnamoyl-CoA, and (N,N-dimethyl-p-amino)cinnamoyl-CoA have been experimentally determined. The carbonyl and beta-carbons are all deshielded, while the alpha-carbons show increased shielding. These chemical shift perturbations are interpreted to suggest that the pi-electrons of the enoyl thiolester are polarized when bound at the active site. Using the crystal structure of (N,N-dimethyl-p-amino)cinnamoyl-CoA bound at the enzyme active site, the shielding tensors were calculated at three different levels of theory, up to a density functional theory model that included all of the contiguous active site residues. These calculations successfully reproduced the observed spectral changes and permitted the electronic polarization of the substrate to be quantified as an electron density difference map. The calculated electron density difference confirms the loss of electrons at the electrophilic beta-carbon and carbonyl carbon, while a slight increase in electron density at the alpha-carbon where proton donation occurs during the hydration reaction and a larger increase in electron density at the carbonyl oxygen are predicted. The energy required to polarize the electrons to the observed extent was calculated to be 3.2 kcal/mol. The force that provides the requisite energy for the polarization is the interaction of the electric field generated by the protein at the enzyme active site with the polarizable electrons of the substrate. Because the induced electronic polarization is along the predicted reaction pathway, the extent of substrate activation by the induced electronic strain is catalytically relevant.

PMID: 11851410 [PubMed - indexed for MEDLINE]



Source: PubMed