Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing (3-indolpropionyl/acryloyl-CoA as chromophoric substrate analogs

Johnson, Jeffrey K.; Wang, Zhi Xin; Srivastava, D. K.

doi:10.1021/bi00158a020

Cited by 39 publications

(150 citation statements)

References 32 publications

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“…The kinetics of this isomerization have been extensively examined by Srivastava and colleagues, following the polarization of the enone Scheme 5 chromophore, indoleacryloyl-CoA (39,53). The evidence presented above shows the sensitivity of this isomerization reaction to the size of the acyl chain.…”

Section: Discussionmentioning

confidence: 99%

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

1998

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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...

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Section: Discussionmentioning

confidence: 99%

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

1998

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“…Among the acyl-CoA dehydrogenase superfamily, the medium chain fatty acyl-CoA dehydrogenase exhibits such a broad substrate specificity that it can tolerate aromatic group substitution at the position of the fatty acyl-CoA substrate. In this context, 3-indolepropionyl-CoA was converted into 3-indoleacryloyl-CoA according to the general mechanism of all flavin-dependent acyl-CoA dehydrogenases (16). As shown in the Scheme 3, the reaction is initiated by abstraction of the relatively acidic ␣-hydrogen as a proton, followed by the direct transfer of the ␤-hydrogen as a hydride to the oxidized flavin cofactor, yielding a trans-enoyl-CoA product (9).…”

Section: Table II Binding Of Various Tryptophan Analogues To L-tryptomentioning

confidence: 99%

L-Tryptophan 2′,3′-Oxidase from Chromobacterium violaceum

Genet

Bénetti

Hammadi

et al. 1995

Journal of Biological Chemistry

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L-Tryptophan 2,3-oxidase, an amino acid ␣,␤-dehydrogenase isolated from Chromobacterium violaceum, catalyzes the formation of a double bond between the C ␣ and C ␤ carbons of various tryptophan derivatives provided that they possess: (i) a L-enantiomeric configuration, (ii) an ␣-carbonyl group, and (iii) an unsubstituted and unmodified indole nucleus. Kinetic parameters were evaluated for a series of tryptophan analogues, providing information on the contribution of each chemical group to substrate binding. The stereochemistry of the dehydro product was determined to be a Zconfiguration from proton nuclear magnetic resonance assignments. No reaction can be observed in the presence of other aromatic ␤-substituted alanyl residues which behave neither as substrates nor as inhibitors and therefore do not compete against this reaction. The enzymatic synthesis of ␣,␤-dehydrotryptophanyl peptides from 5 to 24 residues was successfully achieved without side product formation, irrespective of the position of the tryptophan residue in the amino acid sequence. A reactional mechanism involving a direct ␣,␤-dehydrogenation of the tryptophan side chain is proposed.

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“…It was recognized that the mechanism underlying these large effects is due to tight H-bonds to the ligand thioester carbonyl oxygen. Polarization of specific ligands, due to transfer of charge toward the thioester carbonyl group has been deduced using UV/Vis (18,21,22) and Raman spectroscopy (23)(24)(25). At an early stage of the structure determination (and at a low resolution), it was not clear whether 2′-or 3′-OH of the ribityl group of the FAD was hydrogen bonded to the thioester carbonyl group.…”

mentioning

confidence: 99%

Mechanism of Activation of Acyl-CoA Substrates by Medium Chain Acyl-CoA Dehydrogenase: Interaction of the Thioester Carbonyl with the Flavin Adenine Dinucleotide Ribityl Side Chain

et al. 1998

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The flavin adenine dinucleotide (FAD) cofactor of pig kidney medium-chain specific acylcoenzyme A (CoA) dehydrogenase (MCADH) has been replaced by ribityl-3′-deoxy-FAD and ribityl-2′-deoxy-FAD. 3′-Deoxy-FAD-MCADH has properties very similar to those of native MCADH, indicating that the FAD-ribityl side-chain 3′-OH group does not play any particular role in cofactor binding or catalysis. 2′-Deoxy-FAD-MCADH was characterized using the natural substrate C 8 CoA as well as various substrate and transition-state analogues. Substrate dehydrogenation in 2′-deoxy-FAD-MCADH is ≈1.5 × 10 7 -fold slower than that of native MCADH, indicating that disruption of the hydrogen bond between 2′-OH and substrate thioester carbonyl leads to a substantial transition-state destabilization equivalent to ≈38 kJ mol -1 . The RC-H microscopic pK a of the substrate analogue 3S-C 8 CoA, which undergoes R-deprotonation on binding to MCADH, is lowered from ≈16 in the free state to ≈11 ((0.5) when bound to 2′-deoxy-FAD-MCADH. This compares with a decrease of the same pK a to ≈5 in the complex with unmodified hwtMCADH, which corresponds to a pK shift of ≈11 pK units, i.e., ≈65 kJ mol -1 [Vock, P., Engst, S., Eder, M., and Ghisla, S. (1998) Biochemistry 37, 1848-1860]. The difference of this effect of ≈6 pK units (≈35 kJ mol -1 ) between MCADH and 2′-deoxy-FAD-MCADH is taken as the level of stabilization of the substrate carbanionic species caused by the interaction with the FAD-2′-OH. This energetic parameter derived from the kinetic experiments (stabilization of transition state) is in agreement with those obtained from static experiments (lowering of RC-H microscopic pK a of analogue, i.e., stabilization of anionic transition-state analogue). The contributions of the two single H-bonds involved in substrate activation (Glu376amide-N-H and ribityl-2′-OH) thus appear to behave additively toward the total effect. The crystal structures of native pMCADH and of 2′-deoxy-FAD-MCADH complexed with octanoyl-CoA/octenoyl-CoA show unambiguously that the FAD cofactor and the substrate/product bind in an identical fashion, implying that the observed effects are mainly due to (the absence of) the FAD-ribityl-2′-OH hydrogen bond. The large energy associated with the 2′-OH hydrogen bond interaction is interpreted as resulting from the changes in charge and the increased hydrophobicity induced by binding of lipophilic substrate. This is the first example demonstrating the direct involvement of a flavin cofactor side chain in catalysis.

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Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing (3-indolpropionyl/acryloyl-CoA as chromophoric substrate analogs

Cited by 39 publications

References 32 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

L-Tryptophan 2′,3′-Oxidase from Chromobacterium violaceum

Mechanism of Activation of Acyl-CoA Substrates by Medium Chain Acyl-CoA Dehydrogenase: Interaction of the Thioester Carbonyl with the Flavin Adenine Dinucleotide Ribityl Side Chain

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