Evidence for electrophilic catalysis in the 4-chlorobenzoyl-CoA dehalogenase reaction: UV, Raman, and 13C-NMR spectral studies of dehalogenase complexes of benzoyl-CoA adducts

Taylor, Katherine A.; Liu, Rui Qin; Liang, Po‐Huang; Price, Jack; Dunaway‐Mariano, Debra; Tonge, Peter J.; Clarkson, John; Carey, Paul R.

doi:10.1021/bi00042a020

Cited by 43 publications

(100 citation statements)

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“…These factors taken together suggest the pK a s for 4-HBA-CoA binding to D145S or D145A are in the region of 6.5-7.0. This is approximately 1.5-2 units below that found for free 4-HBA-CoA (6,26) and demonstrates how the active site can manipulate electron density at the 4-position of the bound ligand, which is likely a prerequisite for the aromatic substitution reaction occurring at that point. …”

Section: Resultsmentioning

confidence: 76%

“…An indication that strong electrostatic forces are present in the active site came from absorption, Raman and NMR spectroscopic studies of enzyme complexes involving the product, and separately, a substrate analogue (6). The spectroscopic data showed that electron-polarizing forces can be brought to bear in the active site, which, in the case of the bound product, cause a major rearrangement of the electrons in the benzoyl moiety.…”

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confidence: 99%

“…Recently, we published an analysis of the polarizing forces in the active site, combining Raman spectroscopic data on the bound product, including results on 13 C and 18 O substituted isotopomers of the product, with structural conclusions culled from the X-ray crystallographic studies (9). The spectral characteristics of strong polarization seen for the bound product are a large shift in λ max , from 292 to 370 nm, and a dramatic rearrangement of the Raman normal modes of the benzoyl ring (6,9). The conclusions from the combined Raman and X-ray data were as follows:…”

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confidence: 99%

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

et al. 1999

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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.

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confidence: 76%

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

et al. 1999

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“…4-HBA-CoA and 4-CBACoA were prepared according to Liang et al (1993), 4-MeBA-CoA according to Taylor et al (1995), and 4-HBApantetheine and 4-CBA-pantetheine according to Taylor (1996). Phenylacetyl-CoA, propionyl-CoA, and tiglyl-CoA were purchased from Sigma Chemical Co. 4-CBA-CoA dehalogenase (specific activity ) 1.5 units/mg) was prepared according to the procedure of Chang et al (1992) as modified in Liang et al (1993).…”

Section: Generalmentioning

confidence: 99%

Investigation of Substrate Activation by 4-Chlorobenzoyl-Coenzyme A Dehalogenase

et al. 1997

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4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolysis of 4-CBACoA to 4-hydroxybenzoyl-coenzyme A (4-HBA-CoA), using the carboxylate side chain of aspartate 145 to displace the chloride from C(4) of the benzoyl ring. Previous UV-visible, Raman, and 13 C NMR studies of enzyme-bound substrate analog or product ligand indicated that the environment of the enzyme active site induces a significant reorganization of the benzoyl ring π-electrons. This observation was interpreted as evidence for electrophilic catalysis [Viz. active-site-induced polarization of electron density away from the ring C(4)] [

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“…However, using a Raman difference spectrometer constructed in 1992 (6), red-excited Raman difference data for the product complex were obtained (37). These showed that the active site of dehalogenase can bring about a complete reorganization of the benzoyl group's electrons and, in turn, provided insight into how the difficult chemical step of replacing the -Cl atom on the ring by an -OH is achieved.…”

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confidence: 99%

Raman Spectroscopy, the Sleeping Giant in Structural Biology, Awakes

Carey

1999

Journal of Biological Chemistry

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Principally through the efforts of crystallographers, we are being presented with an ever expanding atomic view of the biological world. Although this brings into focus many questions regarding the mysteries of function, techniques are needed that facilitate the transition in our understanding from structure to function. Raman spectroscopy is one of these; because the Raman effect involves an intimate interplay between atomic positions, electron distribution, and intermolecular forces, it sits at the bridgehead between structure and function. Thus, the Raman technique can answer questions that lie at the heart of issues such as ligand macromolecule recognition and enzymatic catalysis. Raman spectroscopy involves analyzing the scattered photons from a laser beam focused into the sample solution (1). The inelastic scattered photons (the Raman spectrum) provide information on molecular vibrations that, in turn, yield data on molecular conformation and environment. At its most effective, Raman spectroscopy can provide exquisite detail from an important site in a much larger macromolecular complex. Although Raman was first applied to the definition of biological molecules in the 1930s (2), the giant has remained drowsy due the difficulties both in obtaining high quality data and in interpreting those data. Considerable advances have been made in these areas in the past few years, and the giant is stirring! A major goal of this review is to provide biochemists with enough information to determine whether the Raman technique could provide structural insights into their systems. The specific issues addressed are which type of systems are amenable to study and what information could be obtained. Practically, present-day sample requirements are for 20 l of clear solution, where the target molecule is in the 100 -300 M range. Because the number of vibrational modes of a molecule is 3n Ϫ 6, where n is the number of atoms, the complete Raman spectrum of a macromolecule is exceedingly complex. Thus, Raman is most suited to systems where it is possible to focus upon a small region of interest, e.g. a ligandreceptor or enzyme-substrate binding site. Historically, this condition was achieved by using resonance Raman spectroscopy (1) to obtain the intensity-enhanced spectra from chromophores at specific sites in macromolecules. Recent technical advances mean that similar information can now be gleaned from non-chromophoric systems, markedly broadening the application of the technique. The information obtained can be very detailed, exceeding the level of resolution found in x-ray or NMR analyses (3-5). In addition to providing structural data, the Raman spectrum can also reveal changes in the distribution of electrons in a bound ligand and details of active site-ligand interactions, such as hydrogen bonding strengths.Raman spectroscopy is beginning to fulfill its potential to contribute to structural biology because the three roadblocks that impeded its application to biological systems have been all but removed. These were low se...

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Evidence for electrophilic catalysis in the 4-chlorobenzoyl-CoA dehalogenase reaction: UV, Raman, and 13C-NMR spectral studies of dehalogenase complexes of benzoyl-CoA adducts

Cited by 43 publications

References 18 publications

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

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

Investigation of Substrate Activation by 4-Chlorobenzoyl-Coenzyme A Dehalogenase

Raman Spectroscopy, the Sleeping Giant in Structural Biology, Awakes

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