Electronic Rearrangement Induced by Substrate Analog Binding to the Enoyl-CoA Hydratase Active Site: Evidence for Substrate Activation

D’Ordine, Robert L.; Tonge, Peter J.; Carey, Paul R.; Anderson, Vernon E.

doi:10.1021/bi00208a014

Cited by 53 publications

(66 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In particular, the polarization seen in this work shows striking parallels with that observed for the binding of cinnamoyl-CoA analogues to enoyl-CoA hydratase (34,59) and benzoyl-CoA derivatives to 4-chlorobenzoyl-CoA dehalogenase (60,61). Strong, ground-state polarization of the carbonyl was observed via Raman and 13 C NMR with accompanying sizable red shifts in the UV/VIS spectrum of the bound ligands.…”

Section: Discussionsupporting

confidence: 77%

Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase

1998

View full text Add to dashboard Cite

Oxidation of thioester substrates in the medium-chain acyl-CoA dehydrogenase involves R-proton abstraction by the catalytic base, Glu376, with transfer of a -hydride equivalent to the flavin prosthetic group. Polarization of bound acyl-CoA derivatives by the recombinant human liver enzyme has been studied with 4-thia-trans-2-enoyl-CoA analogues. Polarization is maximal at low pH, with an apparent pK of 9.2 for complexes with the C8 analogue, and progressively lower pK values as the length of the chain increases. This pH effect reflects ionization of the catalytic base, since polarization of a variety of enoyl-CoA analogues by the Glu376Gln mutant is pH independent. Binding of these ligands is accompanied by uptake of about 1 proton with the wild-type enzyme, but only about 0.1 proton with the Glu376Gln mutant. Rapid reaction studies show that proton uptake with the wild-type enzyme occurs at the same rate as polarization of the enoyl-CoA thioester, but is much slower than the initial ligand binding step. Studies with 6-OH-FAD-substituted enzyme show that this isomerization reaction also influences the flavin prosthetic group inducing deprotonation to the green anionic form. The failure of the bound thioether analogue, octyl-SCoA, to elicit pK shifts to flavin and Glu376 shows the importance of the thioester carbonyl oxygen in modulating key properties of the medium-chain enzyme. The role of thioester-mediated desolvation within the active site of the acyl-CoA dehydrogenases is discussed.The reductive half-reaction in the acyl-CoA dehydrogenases involves abstraction of the pro-R-R-proton of a bound acyl-CoA thioester with elimination of the pro-R--hydrogen as a hydride equivalent to the N-5 position of the flavin. The reaction appears concerted with normal (Scheme 1) substrates (1-5). The transition state for the dehydrogenation reaction is likely to have appreciable enolate character as negative charge migrates from the carboxylate base through the thioester to the flavin prosthetic group (6-10) (Scheme 2). Substitution of the C-3 methylene group in substrate analogues such as compounds 1 and 2 in Chart 1 precludes hydride transfer, but proton abstraction leads to the generation of strongly absorbing enolate to flavin chargetransfer complexes (7,11,12). These studies illustrate the profound stabilization of the enolates of these weakly acidic substrate analogues (with pK values lowered by 8-12 units; 7, 11, 12) on binding to the enzyme. The acidification of normal thioester substrates has been suggested to be even greater (12). The crystal structure of the medium-chain acylCoA dehydrogenase complexed with acyl-CoA substrates (13) suggests that the developing enolate might be stabilized in part by two hydrogen bonds: one from the 2′-OH of the ribityl side chain of FAD and one from a peptide N-H group to the substrate carbonyl oxygen (7,10,13; heavy dashed lines in Scheme 2). Thus, removal of one of these interactions by reconstitution of the enzyme with 2′-deoxy-FAD leads to a profound (g10 6 -fold) sl...

show abstract

Section: Discussionsupporting

confidence: 77%

Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase

1998

View full text Add to dashboard Cite

show abstract

“…A reasonable explanation is that the protein environment perturbs the electronic properties of the bound HOPDA, as has been reported for several other ligands when bound to proteins. For example, the absorbance maximum of an ␣,␤-unsaturated thiol ester inhibitor was red-shifted 90 nm when bound to the active site of crotonase (20). Similarly, the spectrum of a substituted stilbene derivative was red-shifted 102 nm upon binding to an antibody (21).…”

Section: Discussionmentioning

confidence: 99%

The Tautomeric Half-reaction of BphD, a C-C Bond Hydrolase

Horsman

Bhowmik

Seah

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem- Bacteria use meta-cleavage pathways to degrade a large variety of aromatic compounds, and some alicyclic compounds, such as steroids (1). Although each pathway has its own substrate specificity, they all employ the same underlying logic: vicinal dihydroxylation of an aromatic ring enables dioxygenase-catalyzed extradiol (or meta) ring opening. The resulting meta-cleavage product (MCP) 3 is degraded by an MCP hydrolase, which adds water across a carbon-carbon bond to generate a dienoate and a carboxylic acid (Fig.

show abstract

“…However, the enzyme active site environment may perturb the energy of this transition by polarizing the π electrons of the α,β-unsaturated keto tautomer. For example, a 90 nm red-shift produced by an α,β-unsaturated thiol ester inhibitor when bound to the active site of crotonase was attributed to π electron polarization (47). Analogous polarization of the α,β-unsaturated moiety of ketonized HOPDA may occur in the active site of BphD.…”

Section: Nature Of the Red-shifted Intermediatementioning

confidence: 99%

Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

et al. 2006

View full text Add to dashboard Cite

Kinetic and structural analyses of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) hydrolase from Burkholderia xenovorans LB400 (BphD LB400 ) provide insight into the catalytic mechanism of this unusual serine hydrolase. Single turnover stopped-flow analysis at 25 °C showed that the enzyme rapidly (1/τ 1 ∼ 500 s −1 ) transforms HOPDA (λ max = 434 nm) to a species with electronic absorption maxima at 473 and 492 nm. The absorbance of this enzyme-bound species (E:S) decayed in a biphasic manner (1/τ 2 = 54 s −1 , 1/τ 3 = 6 s −1 ∼ k cat ) with simultaneous biphasic appearance (48 and 8 s −1 ) of an absorbance band at 270 nm characteristic of one of the products, 2-hydroxypenta-2,4-dienoic acid (HPD). Increasing solution viscosity with glycerol slowed 1/τ 1 and 1/τ 2 , but affected neither 1/τ 3 nor k cat , suggesting that 1/τ 2 may reflect diffusive HPD dissociation, while 1/τ 3 represents an intramolecular event. Product inhibition studies suggested that the other product, benzoate, is released after HPD. Contrary to studies in a related hydrolase, we found no evidence that ketonized HOPDA is partially released prior to hydrolysis, and therefore postulate that the biphasic kinetics reflect one of two mechanisms, pending assignment of E:S (λ max = 492 nm). Crystal structures of wild type, the S112C variant, and S112C incubated with HOPDA were each determined to 1.6 Å resolution. The latter reveals interactions between conserved active site residues and the dienoate moiety of the substrate. Most notably, the catalytic residue His265 is hydrogenbonded to the 2-hydroxy/oxo substituent of HOPDA, consistent with a role in catalyzing ketonization. The data are more consistent with an acyl-enzyme mechanism than with the formation of a gem-diol intermediate.The microbial degradation of aromatic compounds is crucial to maintaining the global carbon cycle (1). Aerobic degradation typically involves oxygenation of the aromatic ring to produce a catechol followed by a dioxygenase-catalyzed ring-opening reaction. In one type of degradation pathway, ring-opening yields a meta-cleavage product (MCP) 1 . A serine hydrolase † This work was funded by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant) and the National Institutes of Health (GM-52381).*To whom correspondence should be addressed: Lindsay D. Eltis, leltis@interchange.ubc.ca, Phone: (604) Fax: (604)822 −6041. § These authors contributed equally to this work. ¶ Present addresses: JK: University of Kentucky, Lexington, KY 40506, USA; SD: Howard Hughes Medical Institute, National Jewish Medical and Research Center, Denver, CO 80206; SS: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada 1 Abbreviations: MCP, meta-cleavage product; HPD, 2-hydroxy-penta-2,4-dienoic acid; PCB, polychlorinated biphenyl; HOPDA, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid; DHB, 2,3-dihydroxybiphenyl; DHBD, dihydroxybiphenyl dioxygenase; S k , ketonized substrate; E:S k , enzyme-bound ketonized substrate; P...

show abstract

Electronic Rearrangement Induced by Substrate Analog Binding to the Enoyl-CoA Hydratase Active Site: Evidence for Substrate Activation

Cited by 53 publications

References 25 publications

Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase

Protonic Equilibria in the Reductive Half-Reaction of the Medium-Chain Acyl-CoA Dehydrogenase

The Tautomeric Half-reaction of BphD, a C-C Bond Hydrolase

Kinetic and Structural Insight into the Mechanism of BphD, a C−C Bond Hydrolase from the Biphenyl Degradation Pathway

Contact Info

Product

Resources

About