RAMAN-STUDY OF THE POLARIZING FORCES PROMOTING CATALYSIS IN 4-CHLOROBENZOATE-COA DEHALOGENASE

The enzyme 4-chlorobenzoate-CoA dehalogenase catalyzes the hydrolysis of 4-chlorobenzoate-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-MBA-CoA ). In order to facilitate electrophilic catalysis, the dehalogenase ut ilizes a strong polarizing interaction between the active site residue s and the benzoyl portion of the substrate [Taylor, K. L., et al. (199 5) Biochemistry 34, 13881]. As a result of this interaction, the norma l modes of the benzoyl moiety of the bound 4-HBA-CoA undergo a drastic rearrangement as shown by Raman spectroscopy, Here, we present Raman difference spectroscopic data on the product-enzyme complex where the product's benzoyl carbonyl is labeled with O-18 (C=O-18) Or C-13 (C-13 =O) or where the 4-OH group is labeled with O-18. The data demonstrate that the carbonyl group participates in the most intense normal modes occurring in the Raman spectrum in the 1520-1560 cm(-1) region. The s ubstrate analog 4-methylbenzoate-CoA (4-MeBA-CoA) has also been charac terized by Raman difference spectroscopy in its free form and bound to the dehalogenase, Upon binding, the 4-MeBA-CoA shows evidence of pola rization within the delocalized pi-electrons, but to a lesser extent c ompared to that seen for the product. The use of 4-MeBA-CoA labeled wi th O-18 at the carbonyl enables us to estimate the degree of electron polarization within the C=O group of the bound 4-MeBA-CoA. The C=O str etching frequency occurs near 1663 cm(-1) in non-hydrogen bonding solv ents such as CCl4, near 1650 cm(-1) in aqueous solution, and near 1610 cm(-1) in the active site of dehalogenase, From model studies, we can estimate that in the active site the carbonyl group behaves as though it is being polarized by hydrogen bonds approximately 57 kJ mol(-1) i n strength. Major contributions to this polarization come from hydroge n bonds from the peptide NHs of Gly114 and Phe64. However, an addition al contribution, which may account for up to half of the observed shif t in nu(C=O), originates in the electrostatic field due to the alpha-h elix dipole from residues 121-114, The helix which terminates at Gly11 4, near the C=O group of the bound benzoyl, provides a dipolar electro static component which contributes to the polarization of the C=O bond and to the polarization of the entire benzoyl moiety. The effect of b oth the helix dipole and the hydrogen bonds on the C=O is a ''pull'' o f electrons onto the carbonyl oxygen which, in turn, polarizes the ele ctron distribution within the benzoyl pi-electron system. The ability of these two factors to polarize the electrons within the benzoyl moie ty is increased by the environment about the benzoyl ring; it is surro unded by hydrophobic residues which provide a low-dielectric constant microenvironment. Electron polarization promotes catalysis by reducing electron density at the C4 position of the benzoyl ring, thereby assi sting attack by the side chain of Asp145, An FTTR study on the model c ompound 4-methylbenzoyl S-ethyl thioester, binding to a number of hydr ogen bonding donors in CCl4, is described and is used to relate the ob served shift of the C=O stretching mode of 4-MeBA-CoA in the active si te to the hydrogen bonding strength value, Since the shift of the C=O frequency upon binding is due To hydrogen bonding and helix dipole eff ects, we refer to this bonding strength as the effective hydrogen bond ing strength.