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1.
Antimicrob Agents Chemother ; 56(12): 6284-90, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23006755

ABSTRACT

Tuberculosis (TB) is a global pandemic requiring sustained therapy to facilitate curing and to prevent the emergence of drug resistance. There are few adequate tools to evaluate drug dynamics within infected tissues in vivo. In this report, we evaluated a fluorinated analog of isoniazid (INH), 2-[(18)F]fluoroisonicotinic acid hydrazide (2-[(18)F]-INH), as a probe for imaging Mycobacterium tuberculosis-infected mice by dynamic positron emission tomography (PET). We developed a tail vein catheter system to safely deliver drugs to M. tuberculosis aerosol-infected mice inside sealed biocontainment devices. Imaging was rapid and noninvasive, and it could simultaneously visualize multiple tissues. Dynamic PET imaging demonstrated that 2-[(18)F]-INH was extensively distributed and rapidly accumulated at the sites of infection, including necrotic pulmonary TB lesions. Compared to uninfected animals, M. tuberculosis-infected mice had a significantly higher PET signal within the lungs (P < 0.05) despite similar PET activity in the liver (P > 0.85), suggesting that 2-[(18)F]-INH accumulated at the site of the pulmonary infection. Furthermore, our data indicated that similar to INH, 2-[(18)F]-INH required specific activation and accumulated within the bacterium. Pathogen-specific metabolism makes positron-emitting INH analogs attractive candidates for development into imaging probes with the potential to both detect bacteria and yield pharmacokinetic data in situ. Since PET imaging is currently used clinically, this approach could be translated from preclinical studies to use in humans.


Subject(s)
Hydrazines/pharmacokinetics , Isonicotinic Acids/pharmacokinetics , Mycobacterium tuberculosis , Radiopharmaceuticals/pharmacokinetics , Tuberculosis, Pulmonary/diagnostic imaging , Animals , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Female , Hydrazines/administration & dosage , Injections/methods , Isonicotinic Acids/administration & dosage , Isotope Labeling , Mice , Mice, Inbred BALB C , Positron-Emission Tomography , Radiopharmaceuticals/administration & dosage , Spectrophotometry, Ultraviolet , Tissue Distribution , Tomography, X-Ray Computed , Tuberculosis, Pulmonary/microbiology
2.
Phys Rev Lett ; 90(11): 118103, 2003 Mar 21.
Article in English | MEDLINE | ID: mdl-12688970

ABSTRACT

Photoabsorption studies of red fluorescent protein chromophore anions have been performed at the ELISA electrostatic heavy-ion storage ring. The broad absorption band due to electronic excitation of the chromophores is tuned to a longer wavelength (redshifted) by extending the electronic conjugation of the molecule. A clear vibrational progression is resolved with E(vib) approximately 380 and 520 cm(-1) for two different forms of the chromophore. The vibrational modes correspond to collective motions of the entire molecular structure. It is argued that the excited electronic state has an equilibrium configuration far from that of the electronic ground state, i.e., poor Franck Condon overlap.


Subject(s)
Luminescent Proteins/chemistry , Absorption , Anions , Photochemistry , Spectrophotometry/methods , Static Electricity , Red Fluorescent Protein
4.
Biochemistry ; 40(6): 1725-33, 2001 Feb 13.
Article in English | MEDLINE | ID: mdl-11327833

ABSTRACT

Raman spectroscopy has been used to investigate the structure of a substrate analogue, hexadienoyl-CoA (HD-CoA), bound to wild-type enoyl-CoA hydratase and G141P, a mutant in which a hydrogen bond to the substrate carbonyl has been removed. Raman spectra of isotopically labeled HD-CoAs, together with normal mode calculations, confirm the selective ground-state polarization of the enone fragment previously suggested to occur on binding to the wild-type enzyme [Tonge, P. J., Anderson, V. E., Fausto, R., Kim, M., Pusztai-Carey, M., and Carey, P. R. (1995) Biospectroscopy 1, 387-394]. In addition, Raman spectra of HD-CoA bound to the G141P mutant enzyme demonstrate that the hydrogen bond between the G141 amide NH group and the substrate carbonyl is critical for polarization and activity. Replacement of G141 with proline results in an approximately 10(6)-fold decrease in k(cat) and eliminates the ability of the enzyme to polarize the substrate analogue. As G141 is part of a consensus sequence in the enoyl-CoA hydratase superfamily, the results presented here provide direct evidence for the importance of the oxyanion hole in the reactions catalyzed by other family members.


Subject(s)
Enoyl-CoA Hydratase/metabolism , Glycine/metabolism , Acyl Coenzyme A/chemistry , Acyl Coenzyme A/metabolism , Animals , Binding Sites/genetics , Carbon Isotopes/metabolism , Enoyl-CoA Hydratase/chemistry , Enoyl-CoA Hydratase/genetics , Enzyme Activation/genetics , Glycine/genetics , Hydrogen Bonding , Kinetics , Oxygen Isotopes/metabolism , Proline/genetics , Protein Conformation , Rats , Solutions , Spectrum Analysis, Raman , Substrate Specificity/genetics
5.
Biochemistry ; 40(2): 440-8, 2001 Jan 16.
Article in English | MEDLINE | ID: mdl-11148038

ABSTRACT

Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSOR(mod)D, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other studies. This oxo ligand is found to be exchangeable directly with DMS(18)O or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm(-1) band, an extra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'as prepared' enzyme which is not present in the 'redox cycled' enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSOR(mod)D form, identified by its characteristic Raman spectrum, is also present in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase.


Subject(s)
Molybdenum/chemistry , Oxidoreductases/chemistry , Rhodobacter capsulatus/enzymology , Bacterial Proteins/chemistry , Binding Sites , Iron-Sulfur Proteins/chemistry , Ligands , Oxidation-Reduction , Spectrum Analysis, Raman/methods , Sulfides/chemistry
6.
Biochemistry ; 39(45): 13982-92, 2000 Nov 14.
Article in English | MEDLINE | ID: mdl-11076541

ABSTRACT

2,4-Hexadienoyl-coenzyme A (HD-CoA) has been used to investigate the redox and ionization properties of medium-chain acyl-CoA dehydrogenase (MCAD) from pig kidney. HD-CoA is a thermodynamically stabilized product analogue that binds tightly to oxidized MCAD (K(dox) = 3.5 +/- 0.1 microM, pH 7.6) and elicits a redox potential shift that is 78% of that observed with the natural substrate/product couple [Lenn, N. D., Stankovich, M. T., and Liu, H. (1990) Biochemistry 29, 3709-3715]. The midpoint potential of the MCAD.HD-CoA complex exhibits a pH dependence that is consistent with the redox-linked ionization of two key glutamic acids as well as the flavin adenine dinucleotide (FAD) cofactor. The estimated ionization constants for Glu376-COOH (pK(a,ox) approximately 9.3) and Glu99-COOH (pK(a,ox) approximately 7.4) in the oxidized MCAD.HD-CoA complex indicate that while binding of the C(6) analogue makes Glu376 a stronger catalytic base (pK(a,ox) approximately 6.5, free MCAD), it has little effect on the pK of Glu99 (pK(a,ox) approximately 7.5, free MCAD) [Mancini-Samuelson, G. J., Kieweg, V., Sabaj, K. M., Ghisla, S., and Stankovich, M. T. (1998) Biochemistry 37, 14605-14612]. This finding is in agreement with the apparent pK of 9.2 determined for Glu376 in the human MCAD.4-thia-octenoyl-CoA complex [Rudik, I., Ghisla, S., and Thorpe, C. (1998) Biochemistry 37, 8437-8445]. The pK(a)s estimated for Glu376 and Glu99 in the reduced pig kidney MCAD.HD-CoA complex, 9.8 and 8.6, respectively, suggest that both of these residues remain protonated in the charge-transfer complex under physiological conditions. Polarization of HD-CoA in the enzyme active site may contribute to the observed pK(a) and redox potential shifts. Consequently, the electronic structures of the product analogue in its free and MCAD-bound forms have been characterized by Raman difference spectroscopy. Binding to either the oxidized or reduced enzyme results in localized pi-electron polarization of the hexadienoyl C(1)=O and C(2)=C(3) bonds. The C(4)=C(5) bond, in contrast, is relatively unaffected by binding. These results suggest that, upon binding to MCAD, HD-CoA is selectively polarized such that partial positive charge develops at the C(3)-H region of the ligand, regardless of the oxidation state of the enzyme.


Subject(s)
Acyl Coenzyme A/chemistry , Acyl-CoA Dehydrogenases/chemistry , Acyl Coenzyme A/metabolism , Acyl-CoA Dehydrogenase , Acyl-CoA Dehydrogenases/metabolism , Animals , Binding Sites , Electrochemistry , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Hydrogen-Ion Concentration , Macromolecular Substances , Models, Chemical , Oxidation-Reduction , Potentiometry , Spectrum Analysis, Raman , Swine
7.
Biochemistry ; 39(26): 7645-50, 2000 Jul 04.
Article in English | MEDLINE | ID: mdl-10869170

ABSTRACT

Structural and genetic studies indicate that the antibacterial compound triclosan, an additive in many personal care products, is an inhibitor of EnvM, the enoyl reductase from Escherichia coli. Here we show that triclosan specifically inhibits InhA, the enoyl reductase from Mycobacterium tuberculosis and a target for the antitubercular drug isoniazid. Binding of triclosan to wild-type InhA is uncompetitive with respect to both NADH and trans-2-dodecenoyl-CoA, with K(i)' values of 0.22+/-0.02 and 0.21+/-0.01 microM, respectively. Replacement of Y158, the catalytic tyrosine residue, with Phe, reduces the affinity of triclosan for the enzyme and results in noncompetitive inhibition, with K(i) and K(i)' values of 36+/-5 and 47+/-5 microM, respectively. Consequently, the Y158 hydroxyl group is important for triclosan binding, suggesting that triclosan binds in similar ways to both InhA and EnvM. In addition, the M161V and A124V InhA mutants, which result in resistance of Mycobacterium smegmatis to triclosan, show significantly reduced affinity for triclosan. Inhibition of M161V is noncompetitive with K(i)' = 4.3+/-0.5 microM and K(i) = 4.4+/-0.9 microM, while inhibition of A124V is uncompetitive with K(i)' = 0. 81 +/- 0.11 microM. These data support the hypothesis that the mycobacterial enoyl reductases are targets for triclosan. The M161V and A124V enzymes are also much less sensitive to isoniazid compared to the wild-type enzyme, indicating that triclosan can stimulate the emergence of isoniazid-resistant enoyl reductases. In contrast, I47T and I21V, two InhA mutations that occur in isoniazid-resistant clinical isolates of M. tuberculosis, show unimpaired inhibition by triclosan, with uncompetitive inhibition constants (K(i)') of 0.18+/-0.01 and 0.12+/- 0.01 microM, respectively. The latter result indicates that InhA inhibitors targeted at the enoyl substrate binding site may be effective against existing isoniazid-resistant strains of M. tuberculosis.


Subject(s)
Enzyme Inhibitors/pharmacology , Isoniazid/pharmacology , Mycobacterium tuberculosis/enzymology , Oxidoreductases/antagonists & inhibitors , Triclosan/pharmacology , Antitubercular Agents/pharmacology , Bacterial Proteins , Binding Sites , Drug Resistance, Microbial/genetics , Drug Resistance, Microbial/physiology , Kinetics , Mutation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/metabolism , Oxidoreductases/genetics
8.
Biochemistry ; 39(15): 4423-31, 2000 Apr 18.
Article in English | MEDLINE | ID: mdl-10757992

ABSTRACT

We present Raman spectra, obtained using 752 nm excitation, on wild-type GFP and the S65T mutant of this intrinsically fluorescent protein together with data on a model chromophore, ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate . In the pH range 1-14, the model compound has two macroscopic pK(a)s of 1.8 and 8.2 attributed to ionization of the imidazolinone ring nitrogen and the phenolic hydroxyl group, respectively. Comparison of the model chromophore with the chromophore in wild-type GFP and the S65T mutant reveals that the cationic form, with both the imidazolinone ring nitrogen and the phenolic oxygen protonated, is not present in these particular GFP proteins. Our results do not provide any evidence for the zwitterionic form of the chromophore, with the phenolic group deprotonated and the imidazolinone ring nitrogen protonated, being present in the GFP proteins. In addition, since the position of the Raman bands is a property exclusively of the ground state structure, the data enable us to investigate how protein-chromophore interactions affect the ground state structure of the chromophore without contributions from excited state effects. It is found that the ground state structure of the anionic form of the chromophore, which is most relevant to the fluorescent properties, is strongly dependent on the chromophore environment whereas the neutral form seems to be insensitive. A linear correlation between the absorption properties and the ground state structure is demonstrated by plotting the absorption maxima versus the wavenumber of a Raman band found in the range 1610-1655 cm(-1).


Subject(s)
Acetates/chemistry , Coumaric Acids , Fluorescent Dyes/chemistry , Imidazoles/chemistry , Luminescent Proteins/chemistry , Acetates/chemical synthesis , Acetates/metabolism , Acyl Coenzyme A/chemistry , Acyl Coenzyme A/metabolism , Acyl-CoA Dehydrogenase , Amino Acid Substitution/genetics , Anions/chemistry , Anions/metabolism , Buffers , Cations/chemistry , Cations/metabolism , Fatty Acid Desaturases/metabolism , Fluorescence , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/metabolism , Green Fluorescent Proteins , Humans , Hydrogen-Ion Concentration , Imidazoles/chemical synthesis , Imidazoles/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Mutation/genetics , Nitrogen/metabolism , Oxygen/metabolism , Protein Denaturation , Protons , Solvents , Spectrum Analysis, Raman
10.
Biochemistry ; 39(1): 92-101, 2000 Jan 11.
Article in English | MEDLINE | ID: mdl-10625483

ABSTRACT

4-OH-Cinnamoyl-CoA has been synthesized as a probe of the active site in the medium chain acyl-CoA dehydrogenase. The protonated form of the free ligand (lambda(max) = 336 nm) yields the corresponding phenolate (lambda(max) = 388 nm) with a pK of 8.9. 4-OH-Cinnamoyl-CoA binds tightly (K(d) = 47 nM, pH 6) to the pig kidney dehydrogenase with a prominent new band at 388 nm, suggesting ionization of the bound ligand. However, this spectrum reflects polarization, not deprotonation, of the neutral form of the ligand. Thus, the 388 nm band is abolished as the pH is raised (not lowered), and analogous spectral and pH behavior is observed with the nonionizable analogue 4-methoxycinnamoyl-CoA. Studies with wild type, E99G, and E376Q mutants of the human medium chain acyl-CoA dehydrogenase showed that these two active site carboxylates strongly suppress ionization of the 4-OH ligand. Binding to the double mutant E99G/E376Q gives an intense new band as the pH is raised (pK = 7.8), with an absorbance maximum at 498 nm resembling the natural 4-OH-cinnamoyl-thioester chromophore of the photoactive yellow protein. Raman difference spectroscopy in water and D(2)O, using the free ligand and wild-type and double-mutant enzyme.ligand complexes, confirms that the 4-OH group of the thioester is ionized only when bound to the double mutant. These data demonstrate the strong electrostatic coupling between ligand and enzyme, and the critical role Glu376 plays in modulating thioester polarization in the medium chain acyl-CoA dehydrogenase.


Subject(s)
Acyl Coenzyme A/metabolism , Acyl-CoA Dehydrogenases/metabolism , Coenzyme A/metabolism , Coumaric Acids , Acyl Coenzyme A/chemical synthesis , Acyl-CoA Dehydrogenase , Acyl-CoA Dehydrogenases/chemistry , Acyl-CoA Dehydrogenases/genetics , Animals , Binding Sites , Coenzyme A/chemical synthesis , Esters , Glutamic Acid/genetics , Glutamine/genetics , Glycine/genetics , Humans , Kidney/enzymology , Ligands , Mutagenesis, Site-Directed , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrum Analysis, Raman , Substrate Specificity , Swine
11.
Biochemistry ; 38(41): 13623-34, 1999 Oct 12.
Article in English | MEDLINE | ID: mdl-10521269

ABSTRACT

The role of tyrosine 158 (Y158) and lysine 165 (K165) in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, has been investigated. These residues have been identified as putative catalytic residues on the basis of structural and sequence homology with the short chain alcohol dehydrogenase family of enzymes. Replacement of Y158 with phenylalanine (Y158F) and with alanine (Y158A) results in 24- and 1500-fold decreases in k(cat), respectively, while leaving K(m) for the substrate, trans-2-dodecenoyl-CoA, unaffected. Remarkably, however, replacement of Y158 with serine (Y158S) results in an enzyme with wild-type activity. Kinetic isotope effect studies indicate that the transfer of a solvent-exchangeable proton is partially rate-limiting for the wild-type and Y158S enzymes, but not for the Y158A enzyme. These data indicate that Y158 does not function formally as a proton donor in the reaction but likely functions as an electrophilic catalyst, stabilizing the transition state for hydride transfer by hydrogen bonding to the substrate carbonyl. A conformational change involving rotation of the Y158 side chain upon binding of the enoyl substrate to the enzyme is proposed as an explanation for the inverse solvent isotope effect observed on V/K(DD-CoA) when either NADH or NADD is used as the reductant. These data are consistent with the recently published structure of a C16 fatty acid substrate bound to InhA that shows Y158 hydrogen bonded to the substrate carbonyl group and rotated from the position it occupies in the InhA-NADH binary complex [Rozwarski, D. A., Vilcheze, C., Sugantino, M., Bittman, R., and Sacchettini, J. C. (1999) J. Biol. Chem. 274, 15582-15589]. Finally, the role of K165 has been analyzed using site-directed mutagenesis. Replacement of K165 with glutamine (K165Q) and arginine (K165R) has no effect on the enzyme's catalytic ability or on its ability to bind NADH. However, the K165A and K165M enzymes are unable to bind NADH, indicating that K165 has a primary role in cofactor binding.


Subject(s)
Lysine/chemistry , Mycobacterium tuberculosis/enzymology , Oxidoreductases/chemistry , Tyrosine/chemistry , Amino Acid Substitution/genetics , Bacterial Proteins , Binding Sites/genetics , Catalysis , Circular Dichroism , Deuterium/chemistry , Histidine/genetics , Kinetics , Lysine/genetics , Mutagenesis, Site-Directed , Mycobacterium tuberculosis/genetics , NAD/chemistry , Oxidoreductases/genetics , Oxidoreductases/isolation & purification , Peptide Fragments/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Solvents , Tyrosine/genetics
13.
Biochemistry ; 38(29): 9508-16, 1999 Jul 20.
Article in English | MEDLINE | ID: mdl-10413528

ABSTRACT

The role of two glutamate residues (E164 and E144) in the active site of enoyl-CoA hydratase has been probed by site-directed mutagenesis. The catalytic activity of the E164Q and E144Q mutants has been determined using 3'-dephosphocrotonyl-CoA. Removal of the 3'-phosphate group reduces the affinity of the substrate for the enzyme, thereby facilitating the determination of K(m) and simplifying the analysis of the enzymes' pH dependence. k(cat) for the hydration of 3'-dephosphocrotonyl-CoA is reduced 7700-fold for the E144Q mutant and 630000-fold for the E164Q mutant, while K(m) is unaffected. These results indicate that both glutamate residues play crucial roles in the hydration chemistry catalyzed by the enzyme. Previously, we reported that, in contrast to the wild-type enzyme, the E164Q mutant was unable to exchange the alpha-proton of butyryl-CoA with D(2)O [D'Ordine, R. L., Bahnson, B. J., Tonge, P. J. , and Anderson, V. E. (1994) Biochemistry 33, 14733-14742]. Here we demonstrate that E144Q is also unable to catalyze alpha-proton exchange even though E164, the glutamate that is positioned to abstract the alpha-proton, is intact in the active site. The catalytic function of each residue has been further investigated by exploring the ability of the wild-type and mutant enzymes to eliminate 2-mercaptobenzothiazole from 4-(2-benzothiazole)-4-thiabutanoyl-CoA (BTTB-CoA). As expected, reactivity toward BTTB-CoA is substantially reduced (690-fold) for the E164Q enzyme compared to wild-type. However, E144Q is also less active than wild-type (180-fold) even though elimination of 2-mercaptobenzothiazole (pK(a) 6.8) should require no assistance from an acid catalyst. Clearly, the ability of E164 to function as an acid-base in the active site is affected by mutation of E144 and it is concluded that the two glutamates act in concert to effect catalysis.


Subject(s)
Enoyl-CoA Hydratase/chemistry , Enoyl-CoA Hydratase/metabolism , Glutamic Acid/chemistry , Glutamic Acid/metabolism , Animals , Benzothiazoles , Butyryl-CoA Dehydrogenase , Catalysis , Cinnamates/chemistry , Crotonates/chemistry , Crystallography, X-Ray , Enoyl-CoA Hydratase/genetics , Fatty Acid Desaturases/chemistry , Glutamic Acid/genetics , Hydrogen-Ion Concentration , Imidazoles/chemistry , Kinetics , Mutagenesis, Site-Directed , Phosphorylation , Protein Binding , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Thiazoles/chemistry
14.
J Mol Biol ; 290(4): 859-65, 1999 Jul 23.
Article in English | MEDLINE | ID: mdl-10398587

ABSTRACT

The enoyl-acyl carrier protein reductase (ENR) is involved in bacterial fatty acid biosynthesis and is the target of the antibacterial diazaborine compounds and the front-line antituberculosis drug isoniazid. Recent studies suggest that ENR is also the target for the broad-spectrum biocide triclosan. The 1.75 A crystal structure of EnvM, the ENR from Escherichia coli, in complex with triclosan and NADH reveals that triclosan binds specifically to EnvM. These data provide a molecular mechanism for the antibacterial activity of triclosan and substantiate the hypothesis that its activity results from inhibition of a specific cellular target rather than non-specific disruption of the bacterial cell membrane. This has important implications for the emergence of drug-resistant bacteria, since triclosan is an additive in many personal care products such as toothpastes, mouthwashes and soaps. Based on this structure, rational design of triclosan derivatives is possible which might be effective against recently identified triclosan-resistant bacterial strains.


Subject(s)
Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/chemistry , Oxidoreductases/antagonists & inhibitors , Triclosan/pharmacology , Boron Compounds/pharmacology , Crystallography , Drug Resistance, Microbial , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) , Escherichia coli/enzymology , Escherichia coli Proteins , Fatty Acid Synthase, Type II , Models, Molecular , NAD/chemistry , Oxidoreductases/chemistry , Triclosan/chemistry
15.
Biochemistry ; 36(33): 10192-9, 1997 Aug 19.
Article in English | MEDLINE | ID: mdl-9254617

ABSTRACT

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.


Subject(s)
Escherichia coli/enzymology , Hydrolases/metabolism , Binding Sites , Catalysis , Gas Chromatography-Mass Spectrometry , Hydrolases/chemistry , Oxygen Isotopes , Spectrum Analysis, Raman
16.
Biochemistry ; 36(8): 2211-20, 1997 Feb 25.
Article in English | MEDLINE | ID: mdl-9047322

ABSTRACT

The structure of the substrate analog 2,4-hexadienoyl-coenzyme A (HD-CoA) bound to the enzyme enoyl-CoA hydratase has been determined using transferred nuclear Overhauser enhancement (TRNOE) spectroscopy. NOEs between the adenine H8 proton and several pantetheine protons in the bound form of HD-CoA indicate that the overall structure of the CoA molecule is bent, while NOEs between adenine and ribose protons indicate that the conformation about the glycosidic bond is anti. The absence of long range NOEs along the pantetheine moiety is consistent with this region of the molecule being bound in an extended conformation. In addition, NOEs between the vinylic protons indicate that the HD moiety is s-trans about C3-C4. The conformation of the CoA portion of bound HD-CoA is strikingly similar to that of the CoA portion of 4-(hydroxybenzoyl)-CoA bound to the active site of 4-(chlorobenzoyl)-CoA dehalogenase [Benning, M. M., et al. (1996) Biochemistry 35, 8103-8109]. The structural similarity of the ligands along with the primary sequence homology validates the modeling of the enoyl-CoA hydratase structure with the 4-(chlorobenzoyl)-CoA dehalogenase backbone. The homology modeling allows the prediction that the enoyl-CoA substrates are bound in an s-cis conformation about C1-C2 and that Glu 144 is present at the active site and can function as a general acid/base.


Subject(s)
Alkadienes/chemistry , Coenzyme A/chemistry , Enoyl-CoA Hydratase/chemistry , Hydrolases/chemistry , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Enoyl-CoA Hydratase/metabolism , Hydrolases/metabolism , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Sequence Alignment , Sequence Analysis
17.
Biochemistry ; 35(38): 12487-94, 1996 Sep 24.
Article in English | MEDLINE | ID: mdl-8823184

ABSTRACT

In order to investigate structure-reactivity relationships within a series of acyl cysteine proteases [Doran, J. D., & Carey, P. R. (1996) Biochemistry 35, 12495-12502], deacylation kinetics have been measured for a number of acyl intermediates involving members of the papain superfamily. Derivatives of the "simple" chromophoric ligand (5-methylthienyl)acrylate (5MTA) and those based on two chromophorically labeled derivatives of peptidyl substrates, viz., 2-[(N-acetyl-L-phenylalanyl)amino]-3-(5-methylthienyl)acrylate (Phe5MTA) and 2-[(N-acetyl-L-alanyl)amino]-3-(5-methylthienyl)acrylate (Ala5MTA), were used to create acyl enzyme adducts with papain, cathepsin B, and cathepsin L. The chromophoric specific substrates were designed to utilize hydrogen-bonding and hydrophobic interactions which are known to be important in promoting catalysis by papain. For cathepsins B and L, removing one of the hydrogen-bonding donors making up the putative oxyanion hole retards deacylation by 3-25-fold, demonstrating that the oxyanion hole has a modest effect on catalysis for these substrates. With the above substrates and the wild-type and oxyanion hole mutants, the values of the deacylation rate constants stretch over a 214-fold range, from 0.07 to 15 x 10(-3) s-1. The pKa for deacylation of [(5-methylthienyl)-acryloyl]papain is 4.9, close to that reported for similar papain intermediates, while that for Ala5MTA-papain is at 3.5, which in the latter likely represents the effect of the P1-S1 and P2-S2 interactions on the environment of histidine-159. For the Phe5MTA-papain the extent of deacylation was found to depend on the pH and the starting acyl enzyme concentration. A simple model has been derived which accounts quantitatively for this behavior, using the assumptions that the protonated form of the acyl product reacylates the enzyme and that in the pH range 5.0-7.5 the ionization of active-site groups has no effect on reacylation. The validity of the first assumption was demonstrated by following the deacylation of Phe5MTA-papain in the presence of the potent inhibitor E-64 [1-(L-trans-epoxysuccinyl-L-leucylamino)-4-guanidinobutane], whereupon complete deacylation occurred at all pHs with a pKa identical to that for Ala5MTA-papain, viz., 3.5.


Subject(s)
Cysteine Endopeptidases/metabolism , Endopeptidases , Papain/metabolism , Acrylates/metabolism , Acylation , Animals , Cathepsin B/metabolism , Cathepsin L , Cathepsins/metabolism , Escherichia coli/genetics , Esters/metabolism , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Molecular Structure , Mutagenesis, Site-Directed , Rats , Structure-Activity Relationship
18.
Biochemistry ; 34(42): 13881-8, 1995 Oct 24.
Article in English | MEDLINE | ID: mdl-7577982

ABSTRACT

This paper reports on the mechanism of substrate activation by the enzyme 4-chlorobenzoyl coenzyme A dehalogenase. This enzyme catalyzes the hydrolytic dehalogenation of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) to 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 Meisenheimer complex. Loss of chloride ion from this intermediate results in the formation of an arylated enzyme intermediate. The arylated enzyme is hydrolyzed to free enzyme plus 4-HBA-CoA by the addition of water 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 site carboxylate group. UV-visible, 13C-NMR, and Raman spectroscopic techniques were used to monitor changes in the distribution of the pi electrons 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 dehalogenase active site. The UV-visible spectra measured for 4-HBA-CoA in aqueous buffer at pH 7.5 and in the dehalogenase active site revealed that a large red shift (from 292 to 373 nm) in the lambda max of the benzoyl moiety occurs upon binding.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Acyl Coenzyme A/metabolism , Hydrolases/metabolism , Pseudomonas/enzymology , Acyl Coenzyme A/chemistry , Alcohol Dehydrogenase/chemistry , Alcohol Dehydrogenase/metabolism , Binding Sites , Enoyl-CoA Hydratase/chemistry , Enoyl-CoA Hydratase/metabolism , Hydrolases/chemistry , Magnetic Resonance Spectroscopy , Molecular Structure , Spectrophotometry , Spectrum Analysis, Raman , Steroid Isomerases/chemistry , Steroid Isomerases/metabolism
19.
Biochemistry ; 33(49): 14733-42, 1994 Dec 13.
Article in English | MEDLINE | ID: mdl-7993901

ABSTRACT

3-Quinuclidinone catalyzes the exchange of the alpha-protons of butyryl-coenzyme A (CoA) with a second-order rate constant of 2.4 x 10(-6) M-1 s-1. In contrast, enoyl-CoA hydratase catalyzes the stereospecific exchange of the pro-2S proton of butyryl-CoA with a maximum second-order rate constant of ca. 8 x 10(2) M-1 s-1. This isotope exchange reaction is completely stereospecific within the limits of experimental detection (over 600-fold). The enzyme-catalyzed exchange is dependent on pD, decreasing above a pKa of 8.8 and below a pKa of 8.1, but independent of the buffer concentration. The stereospecificity of the exchange was unexpected because the pro-2R hydrogen is abstracted during the enzyme-catalyzed dehydration of 3(S)-hydroxybutyryl-CoA. In spite of the ability to exchange the pro-2S hydrogen, the stereospecificity of the dehydration reaction was determined to be better than 1 in 10(5) as no incorporation of 2H into the alpha-position of crotonyl-CoA or into the pro-2S position of 3(S)-hydroxybutyryl-CoA was detected during prolonged equilibrations with enoyl-CoA hydratase. Both the exchange of the alpha-proton and the dehydration activity of the enzyme are diminished by over 100-fold in a site-directed mutation of rat liver enoyl-CoA hydratase, where glutamate-164 is changed to glutamine, strongly suggesting that the same active site base is responsible for proton abstraction in both the dehydration and solvent exchange reactions. The enoyl-CoA hydratase-catalyzed exchange of the alpha-protons becomes nonstereospecific when the acidity of the alpha-protons is enhanced. While alpha-proton abstraction can be observed when no elimination reaction is possible, there is no evidence for proton abstraction without elimination in the crotonase equilibrations with 3(S)-hydroxybutyryl-CoA, 3-hydroxypropionyl-CoA, or 3-chloropropionyl-CoA. The differences in the isotope exchange and dehydration reactions emphasize the importance of the 3-hydroxyl group in promoting elimination and are consistent with a concerted elimination mechanism.


Subject(s)
Acyl Coenzyme A/metabolism , Enoyl-CoA Hydratase/metabolism , Sulfhydryl Compounds/metabolism , Animals , Catalysis , Cattle , Esters/metabolism , Liver/metabolism , Magnetic Resonance Spectroscopy , Models, Chemical , Protons , Quinuclidines/metabolism , Rats , Stereoisomerism
20.
Biochemistry ; 33(42): 12635-43, 1994 Oct 25.
Article in English | MEDLINE | ID: mdl-7918489

ABSTRACT

A series of alpha,beta unsaturated CoA thiol esters have been characterized spectroscopically when they form noncovalent complexes at the active site of enoyl-CoA hydratase. The UV spectra of all of the thiol esters display significant red shifts when the esters are bound to the crotonase active site. The red shift increases with the ability of a para substituent of substituted cinnamoyl-CoA thiol esters to donate electrons by resonance. The affinity of the substituted cinnamoyl-CoA thiol esters is enhanced by electron-donating substituents, with the slope of the log of the ratio of the inhibition constants versus sigma p+ being near unity. Affinity is also increased by either para or meta electron-withdrawing substituents, suggesting that the enzyme stabilizes a partial positive charge at C-3. Binding to crotonase was shown to decrease the shielding of [3-13C,3-2H]cinnamoyl-CoA by +3.2 ppm, consistent with an increased partial positive charge at C-3. The Raman spectra of cinnamoyl-CoA bound at the crotonase active site similarly reflect the significant electronic ground state changes in the pi electronic structure of the bound substrate. These data show that a major rearrangement of electrons occurs in the acryloyl portion of the cinnamoyl group upon binding, while only a minor perturbation occurs to the distribution of electrons in the phenyl ring.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Acyl Coenzyme A/metabolism , Enoyl-CoA Hydratase/metabolism , Binding Sites , Chromatography, High Pressure Liquid , Electrochemistry , Kinetics , Magnetic Resonance Spectroscopy , Metals/metabolism , Models, Chemical , Protein Binding , Spectrophotometry , Spectrum Analysis, Raman
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