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-HBA-CoA). In order to facilitate electrophilic catalysis, the dehalogenase utilizes a strong polarizing interaction between the active site residues and the benzoyl portion of the substrate [Taylor, K. L., et al. (1995) Biochemistry 34, 13881]. As a result of this interaction, the normal 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 18O (C=18O) or 13C (13C=O) or where the 4-OH group is labeled with 18O. 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 substrate analog 4-methylbenzoate-CoA (4-MeBA-CoA) has also been characterized by Raman difference spectroscopy in its free form and bound to the dehalogenase. Upon binding, the 4-MeBA-CoA shows evidence of polarization within the delocalized pi-electrons, but to a lesser extent compared to that seen for the product. The use of 4-MeBA-CoA labeled with 18O 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 stretching frequency occurs near 1663 cm-1 in non-hydrogen bonding solvents 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 in strength. Major contributions to this polarization come from hydrogen bonds from the peptide NHs of Gly114 and Phe64. However, an additional contribution, which may account for up to half of the observed shift in nuC=O, originates in the electrostatic field due to the alpha-helix dipole from residues 121-114. The helix which terminates at Gly114, near the C=O group of the bound benzoyl, provides a dipolar electrostatic component which contributes to the polarization of the C=O bond and to the polarization of the entire benzoyl moiety. The effect of both the helix dipole and the hydrogen bonds on the C=O is a "pull" of electrons onto the carbonyl oxygen, which, in turn, polarizes the electron distribution within the benzoyl pi-electron system. The ability of these two factors to polarize the electrons within the benzoyl moiety is increased by the environment about the benzoyl ring; it is surrounded 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 assisting attack by the side chain of Asp145. An FTIR study on the model compound 4-methylbenzoyl S-ethyl thioester, binding to a number of hydrogen bonding donors in CCl4, is described and is used to relate the observed shift of the C=O stretching mode of 4-MeBA-CoA in the active site to the hydrogen bonding strength value. Since the shift of the C=O frequency upon binding is due to hydrogen bonding and helix dipole effects, we refer to this bonding strength as the effective hydrogen bonding strength.
Clarkson
J
J
Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
Tonge
P J
PJ
Taylor
K L
KL
Dunaway-Mariano
D
D
Carey
P R
PR
D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology*; D008401:Gas Chromatography-Mass Spectrometry; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010103:Oxygen Isotopes; D013059:Spectrum Analysis, Raman
D010103:Oxygen Isotopes; D006867:Hydrolases; C084333:4-chlorobenzoyl coenzyme A dehalogenase