Spectroscopic investigations of enzymes: Evaluations of electrostatic effects

Raman, 1H and 19F NMR spectra of substituted cinnamoyl CoA thiolesters bound to enoyl CoA hydratase have been recorded and analyzed with the aim of understanding the source and role of electronic polarization in the hydration of alpha,beta unsaturated CoA thiolester substrates. Raman spectra of several enzyme-bound para-substituted cinnamoyl CoAs demonstrate that acryloyl stretching frequencies for all ligands become red shifted when moving from solution to the enzyme active site. The spectra further reveal that regardless of the electronic nature of the substituent, changes in the acryloyl stretching frequencies remain constant at 35 +/- 7 cm-1. The finding suggests that the observed polarization activates the substrate towards hydration across the alpha,beta ethylenic moiety by two distinct methods: the loss of electron density at the beta position and the reduction of the energy of the lowest unoccupied molecular orbital. The two modes combine to activate the beta carbon to hydroxylation during the hydration reaction.; An examination of the crystal structure-substrate complex1 reveals an alpha-helix whose dipole is directed towards the acryloyl moiety of the bound thiolester. Electrostatic calculations provide evidence of an electric field in excess of 107 V cm-1 within the enzyme active site. The field is directed along the alpha,beta double bond of bound substrates, falling off sharply with distance from the acryloyl moiety of the ligand. 19F NMR spectra acquired of fluorine substituted cinnamoyl CoA molecules confirm the steep electric field gradient predicted by the model.; Two peptidyl amide hydrogen bond donors are positioned within hydrogen bonding distance of the ligand thiotester carbonyl. The results of 1H NMR and quantum chemical computations suggest that hydrogen bond formation stabilizes carbonyl polarization and developing negative charge during the reaction. The combined results that polarization and activation of thiolester substrates towards hydration/dehydration originates in the large electric field within the active site of the enzyme.; 13C NMR and infrared spectra, combined with quantum chemical calculations of deuterium isotope effects suggest a mechanism for the estimation of hydrogen bond strengths within an enzyme active site. Isotope effects on the alpha protons of primary and secondary alcohols are linearly correlated with the enthalpy of hydrogen bond formation in biomolecular complexes. Because the effect is greatest on protons anti to the hydroxyl donor, a plausible model involving increased orbital overlap of nonbonded oxygen electrons with the C-H antibonding orbitals in a 'hyperconjugative' manner is proposed to explain the experimental observations.; 1Brian J. Bahnson; University of Delaware, Newark, DE. Submitted to the Protein Data Bank.