EVIDENCE FOR ELECTROPHILIC CATALYSIS IN THE 4-CHLOROBENZOYL-COA DEHALOGENASE REACTION - UV, RAMAN, AND C-13-NMR SPECTRAL STUDIES OF DEHALOGENASE COMPLEXES OF BENZOYL-COA ADDUCTS

This paper reports on the mechanism of substrate activation by the enz yme 4-chlorobenzoyl coenzyme A dehalogenase. This enzyme catalyzes the hydrolytic dehalogenation of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) t o form 4-hydroxybenzoyl coenzyme A (4-HBA-CoA). The mechanism of this reaction is known to involve attack of an active site carboxylate (Asp or Glu side chain) at C(4) of the substrate benzoyl ring to form a Me isenheimer complex. Loss of chloride ion from this intermediate result s in the formation of an arylated enzyme intermediate. The arylated en zyme is hydrolyzed to free enzyme plus 4-HBA-CoA by the addition of wa ter at the acyl carbon [Yang, G., Liang, P.-H., & Dunaway-Mariano, D. (1994) Biochemistry 33, 8527]. The present studies have focused on the activation of the 4-CBA-CoA for nucleophilic attack by the active sit e carboxylate group. UV-visible, C-13-NMR, and Raman spectroscopic tec hniques were used to monitor changes in the distribution of the pi ele ctrons of the benzoyl moiety of benzoyl-CoA adducts [substituted at C( 4) with methyl (4-MeBA-CoA), methoxy (4-MeOBA-CoA), or hydroxyl (4-HBA -CoA) groups or at C(2) or C(3) with a hydroxyl group (2-HBA-CoA and 3 -HBA-CoA)] resulting from the binding of these ligands to the dehaloge nase active site. The UV-visible spectra measured for 4-HBA-CoA in aqu eous buffer at pH 7.5 and in the dehalogenase active site revealed tha t a large red shift (from 292 to 373 nm) in the lambda(max) of the ben zoyl moiety occurs upon binding. The UV-visible spectra of 4-MeBA-CoA and 4-MeOBA-CoA in aqueous buffer show lambda(max) values for the benz oyl chromophore at ca. 260 nm (epsilon = 4 mM(-1) cm(-1)) and at 292 n m (epsilon = 11 mM(-1) cm(-1)), respectively, which are shifted to 302 nm (epsilon = 6 mM(-1) cm(-1)) and to 323 nm (epsilon = 10 mM(-1) cm( -1)) upon enzyme complexation. In contrast, the other benzoyl-CoA addu cts displayed essentially the same spectral properties on and off the enzyme. The C-13-NMR spectrum was measured for [C-13=0]-4-HBA-CoA (enr iched with the carbon-13 isotope at the thioester C=O) in aqueous buff er and in the enzyme active site. A 2.8 ppm downfield shift of the C-1 3=O resonance was observed upon binding to the enzyme. The Raman spect ra of 4-HBA-CoA and 4-MeBA-CoA bound to the dehalogenase active site p rovide evidence for polarization of the benzoyl pi electrons. Specific ally, the thioester C=O stretch observed at 1646 cm(-1) and the in-pla ne phenyl C-H stretches observed at 1221 and 1173 cm-L in the spectrum of 4-HBA-CoA in aqueous buffer are not discernible in the spectrum of the enzyme-bound 4-HBA-CoA, and the benzene ring modes 8a and 8b obse rved at 1603 and 1589 cm(-1) are replaced by features at 1560 and 1525 cm(-1), respectively. The aromatic ring-stretching modes observed for 4-MeBA-CoA at 1609 and 1581 cm(-1) in buffer undergo shifts to 1596 a nd 1570 cm(-1) upon enzyme complexation, while the thioester C=O stret ch band observed at 1651 cm(-1) (buffer) is shifted to 1610 cm(-1) (en zyme). The UV-visible absorption, C-13-NMR, and Raman spectral data ar e interpreted as evidence for substrate activation via polarization of the benzoyl pi electrons away from C(4) and onto the thioester carbon yl oxygen.