Deacylation and Reacylation for a Series of Acyl Cysteine Proteases, Including Acyl Groups Derived from Novel Chromophoric Substrates

Doran, J.; Tonge, Peter J.; Mort, John S.; Carey, Paul R.

doi:10.1021/bi960648h

Cited by 8 publications

(13 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Gly114 is the N-terminal residue of R-helix 114-121 whose positive pole is directed at the benzoyl CdO. The two amide backbone H-bonds plus a possible contribution from the helix dipole constitutes a sizable electron-withdrawing force (Hol, 1985;Aqvist et al, 1991;Baker & Hubbard, 1984;Sali et al, 1988;Nicholson et al, 1991;Doran et al, 1996;Doran & Carey, 1996). The significance of these interactions in dehalogenase catalysis is underscored by the conservation of the Phe64 loop and Gly114 R-helix among the three known dehalogenase sequences (Lai, 1996) and the sequences of the enzymes of the enoyl-CoA family (Babbitt et al, 1992;DunawayMariano & Babbitt, 1994;Muller-Newen et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 1997

View full text Add to dashboard Cite

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)] [

show abstract

Section: Discussionmentioning

confidence: 99%

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

et al. 1997

View full text Add to dashboard Cite

show abstract

“…4-[ 18 O]HBA-CoA. Solid 4-CBA-CoA (10 mg) was dissolved in 1 mL of H 2 18 O (99.2%) and converted to 4-HBA-CoA by the addition of 31 µL of 4-CBA-CoA dehalogenase (in buffered H 2 16 O, 3.7 mg/mL; specific activity ) 1.5 units/ mg) to give 11.4 mM 4-CBA-CoA and 10 µM 4-CBA-CoA dehalogenase in 5 mM K + Hepes (pH 7.0) (96% H 2 18 O). After 6 h at 25 °C, the 4-[ 18 O]HBA-CoA was purified by gel filtration chromatography (Sephadex G-25 column, 110 × 2.2 cm) using H 2 O as an eluant.…”

Section: Methodsmentioning

confidence: 99%

“…It is also recognized that the field can bring about a red shift in the absorption spectrum of a chromophoric group bound near the R-helix's N terminus (12,13). In addition, it has been suggested that R-helix dipoles can contribute to catalytic rate enhancement (14), and recent Raman and absorption spectroscopic studies on a series of acyl cysteine proteases (15,16) have provided quantitative support for this notion. When chromophoric acyl groups bind to cysteine proteases, substantial red shifts, of up to 60 nm, occur, which can be accompanied by major changes in the acyl group's Raman spectrum (12).…”

mentioning

confidence: 99%

Raman Study of the Polarizing Forces Promoting Catalysis in 4-Chlorobenzoate-CoA Dehalogenase

et al. 1997

Self Cite

View full text Add to dashboard Cite

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 ...

show abstract

“…The Raman difference spectra for acyl cysteine proteases, supported by absorption spectral data, provided different insights from those uncovered for the serine analogs (11,35). Principally, these involve so-called -electron polarization; in the cysteine protease active sites there are strong electrostatic forces that bring about a major rearrangement of the -electrons in the acyl group.…”

Section: Evolving Technology Increases Applications In Enzymologymentioning

confidence: 99%

“…For this reason the ligand modes dominate the difference spectrum. When experiments involve ligands that scatter less intensely, protein features appear in the difference spectrum in both the positive and negative directions, and these are a source of information on changes in protein structure, although the interpretation of many of these features is still in its infancy (33, 34).The Raman difference spectra for acyl cysteine proteases, supported by absorption spectral data, provided different insights from those uncovered for the serine analogs (11,35). Principally, these involve so-called -electron polarization; in the cysteine protease active sites there are strong electrostatic forces that bring about a major rearrangement of the -electrons in the acyl group.…”

mentioning

confidence: 99%

Raman Spectroscopy, the Sleeping Giant in Structural Biology, Awakes

Carey

1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Deacylation and Reacylation for a Series of Acyl Cysteine Proteases, Including Acyl Groups Derived from Novel Chromophoric Substrates

Cited by 8 publications

References 32 publications

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

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

Raman Study of the Polarizing Forces Promoting Catalysis in 4-Chlorobenzoate-CoA Dehalogenase

Raman Spectroscopy, the Sleeping Giant in Structural Biology, Awakes

Contact Info

Product

Resources

About