Modulating Electron Density in the Bound Product, 4-Hydroxybenzoyl-CoA, by Mutations in 4-Chlorobenzoyl-CoA Dehalogenase Near the 4-Hydroxy Group

Abstract

The enzyme 4-chlorobenzoyl-CoA dehalogenase hydrolyzes 4-chlorobenzoyl-CoA (4-CBACoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). Biochemical and crystallographic studies have identified a critical role for the dehalogenase residue Asp 145 in close proximity to the ligand's 4-hydroxy group in the structure of the product-enzyme complex. In the present study the effects of site selective mutations at Asp 145 on the product complex are explored by Raman spectroscopy. The spectral signatures of the WT-product complex, the large red shift in λ max , and the complete reorganization of the benzoyl ring modes in Raman data are absent for the D145E complex. The major spectral perturbations in the WT complex are brought about by strong electron "pull" at the benzoyl carbonyl and electron "push" by the side chain of Asp 145 near the 4-OH group. Acting in concert, these factors polarize the benzoyl's π-electrons. Since the Raman data show that very strong electron pull occurs at the benzoyl's carbonyl in the D145E complex, it is apparent that the needed electron push near the benzoyl's 4-OH group is missing. Thus, very precise positioning of Asp 145's side chain near the benzoyl's 4-position is needed to bring about the dramatic electron reorganization seen in the WT complex, and this criterion cannot be met by the glutamate side chain with its additional CH 2 group. For two other Asp145 mutants D145A and D145S that lack catalytic activity, Raman difference spectroscopic data for product complexes demonstrate the presence of a population of ionized product (i.e., 4-O -) in the active sites. The presence of the ionized phenolate form explains the observation that these complexes have highly red-shifted absorbance maxima with λ maxs near 400 nm. For the WT complex only the 4-OH form is seen, ionization being energetically expensive with the presence of the proximal negative charge on the Asp 145 side chain. Semiquantitative estimates of the pK a for the bound product in D145S and D145A indicate that this ionization lies in the pH 6.5-7.0 range. This is approximately 2 pH units below the pK a for the free product. The Raman spectrum of 4-dimethylaminobenzoyl-CoA undergoes major changes upon binding to dehalogenase. The bound form has two features near 1562 and 1529 cm -1 and therefore closely resembles the spectrum of product bound to wild-type enzyme, which underlines the quinonoid nature in these complexes. The use of a newly developed Raman system allowed us to obtain normal (nonresonance) Raman data for the dehalogenase complexes in the 100-300 µM range and heralds an important advance in the application of Raman spectroscopy to dilute solutions of macromolecules.

The active site dynamics of 4-chlorobenzoyl-CoA dehalogenase

“…4-HBA-CoA in solution has a max at 292 nm and a shoulder near 335 nm. In the mutant D145E, only the 330-nm band is present, and the enzyme has reduced activity (22). The pair of bands in the WT enzyme leads to the conclusion that at least two conformations exist for the side chain of Asp-145 in the unmodified dehalogenase.…”

“…The precise positioning of Asp-145 is critical in the dehalogenation reaction. In addition to the W137F mutant's reduced ability to form the Meisenheimer intermediate, the mutant D145E dehalogenase has a k cat that is 600 times lower than that of the WT enzyme (22). The simple addition of an extra carbon to the nucleophilic side chain has a significant effect on catalysis.…”

Computational Studies on Nonenzymatic and Enzymatic Pyridoxal Phosphate Catalyzed Decarboxylations of 2-Aminoisobutyrate

“…This assumes that the substrate carboxylate group and active-site side-chain functional groups are ionized, as found spectroscopically for functional groups in other enzymes (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). The pyruvoyl dependent histidine decarboxylase does place its substrate carboxylate in a nonpolar environment (19), and nonpolar binding sites were successfully employed in the generation of PLP-dependent catalytic antibodies (20).…”

Raman Spectroscopy, the Sleeping Giant in Structural Biology, Awakes

“…However, in 1997 we were able to modify a commercial spectrograph to optimize its performance for dilute aqueous solutions (7). With this instrument concentration requirements were lowered to 100 -300 M. Collecting data for dehalogenase complexes reached a success rate of 100%, and high quality spectra were obtained for several dozen complexes with different substrate analogs and protein-engineered forms of the enzyme (39). One of these complexes had the surprising property of evolving with time (40).…”

“…However, this population decays rapidly with time. There is also evidence at early times for peaks at 1543 and 1490 cm Ϫ1 , and detailed analysis (39) shows that these are because of the ionized (4-O Ϫ ) form of the product bound to the Asn-145 enzyme. After 5 min the spectra cease evolving, and the signature (Fig.…”