Recombinant Human Liver Medium-Chain Acyl-CoA Dehydrogenase: Purification, Characterization, and the Mechanism of Interactions with Functionally Diverse C8-CoA Molecules

Kl, Peterson; Ee, Sergienko; Wu, Yao‐Wen; Nr, Kumar; Aw, Strauss; Ae, Oleson; Ww, Muhonen; Jb, Shabb; Dk, Srivastava

doi:10.1021/bi00045a039

Cited by 27 publications

(108 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Both enzymes show a similar chain length dependent decrease in the pK app going from 4-thia-C8 to 4-thia-C13 (Table 1; 9). Thus, these data are consistent with the close enzymological and structural similarities between these medium-chain enzymes (7,22,33,37; this work). Scheme 3 FIGURE 1: pH dependence of the polarization of 4-thia-trans-2-dodecenoyl-CoA bound to human wild-type medium-chain acylCoA dehydrogenase.…”

Section: -Thia-trans-2-enoyl-coa Analogues: Active Site Probessupporting

confidence: 84%

“…Since all prior work with the 4-thia-enoyl-CoA ligands was done with enzyme isolated from pig kidney, and because of the minor differences in chain length specificity between the porcine and human enzymes (7,9,22,33,37), it was first necessary to repeat key control experiments with the recombinant human protein. Figure 1 shows a pH titration of the human wild-type medium-chain acyl-CoA dehydrogenase complexed with 0.87 equiv of 4-thia-trans-2-dodecenoyl-CoA.…”

Section: -Thia-trans-2-enoyl-coa Analogues: Active Site Probesmentioning

confidence: 99%

See 1 more Smart Citation

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: -Thia-trans-2-enoyl-coa Analogues: Active Site Probessupporting

confidence: 84%

Section: -Thia-trans-2-enoyl-coa Analogues: Active Site Probesmentioning

confidence: 99%

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

1998

View full text Add to dashboard Cite

show abstract

“…The dissociation constant determined by the BIAcore solution affinity assay of human wild type SBCAD with its product, tigyl-CoA, was 0.47 Ϯ 0.01 M, very close to its affinity for substrate (S)-2-methylbutyryl-CoA. This is also seen with other ACDs with their product (62).…”

Section: Surface Plasmon Resonance Analysis Of the Interactions Of Sbmentioning

confidence: 52%

“…It is notable that the overall K D values of V104L/F105L and F105L toward (S)-2-methybutyryl-CoA as measured with the BIAcore assay were higher than for wild type SBCADs, whereas the K D values of rat and human SBCADs toward (S)-2-methybutyryl-CoA were very close to each other (Table IV). Previous studies have shown that the reductive half-reaction is the rate-limiting step in the ACD dehydrogenation reaction (Scheme 1) when an acyl-CoA dehydrogenase reacts with its optimum substrate (61,62). Because both the V104L/ F105L and F105L mutants exhibit stabilization of the reduced flavin ring of FAD and increased catalytic rates for the overall reaction, the turnover rate for these two mutants of E ox S to I should remain at least as fast as in the wild type enzyme (Table II).…”

Section: Surface Plasmon Resonance Analysis Of the Interactions Of Sbmentioning

confidence: 99%

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Burghardt

Vockley

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

Rat and human short/branched chain acyl-CoA dehydrogenases exhibit key differences in substrate specificity despite an overall amino acid identity of 85% between them. Rat short/branched chain acyl-CoA dehydrogenases (SBCAD) are more active toward substrates with longer carbon side chains than human SBCAD, whereas the human enzyme utilizes substrates with longer primary carbon chains. The mechanism underlying this difference in substrate specificity was investigated with a novel surface plasmon resonance assay combined with absorbance and circular dichroism spectroscopy, and kinetics analysis of wild type SBCADs and mutants with altered amino acid residues in the substrate binding pocket. Results show that a relatively few amino acid residues are critical for determining the difference in substrate specificity seen between the human and rat enzymes and that alteration of these residues influences different portions of the enzyme mechanism. Molecular modeling of the SBCAD structure suggests that position 104 at the bottom of the substrate binding pocket is important in determining the length of the primary carbon chain that can be accommodated. Conformational changes caused by alteration of residues at positions 105 and 177 directly affect the rate of electron transfer in the dehydrogenation reactions, and are likely transmitted from the bottom of the substrate binding pocket to ␤-sheet 3. Differences between the rat and human enzyme at positions 383, 222, and 220 alter substrate specificity without affecting substrate binding. Modeling predicts that these residues combine to determine the distance between the flavin ring of FAD and the catalytic base, without changing the opening of the substrate binding pocket.The acyl-CoA dehydrogenases (ACDs) 1 are a family of related enzymes that catalyze the ␣, ␤-dehydrogenation of acylCoA esters, transferring electrons to electron transferring flavoprotein (1-4). Deficiencies of these enzymes are important causes of human disease (3-7). Biochemical and immunological studies have identified at least 9 distinct members of this enzyme family, each having a characteristic substrate utilization pattern (8 -13). Very long, long, medium, and short chain acyl-CoA dehydrogenases (VLCAD, LCAD, MCAD, and SCAD, respectively) catalyze the first step in the mitochondrial ␤-oxidation of fatty acids with substrate optima of 16, 16, 8, and 4 carbon chains, respectively (3, 14 -15). A new ACD has been identified recently (ACAD9) that is also active against long chain acyl-CoA substrates (16). Isovaleryl-CoA dehydrogenase (IVD), short/branched chain acyl-CoA dehydrogenase (SBCAD, also known as 2-methyl branched chain acyl-CoA dehydrogenase), and isobutyryl-CoA dehydrogenase (IBD) catalyze the third step in leucine, isoleucine, and valine metabolism, respectively (2-4, 11, 12, 17, 18), whereas glutaryl-CoA dehydrogenase is active in lysine metabolism (19).We have shown previously that SCAD, SBCAD, and IBD can all utilize butyryl-CoA as substrate (albeit with different efficiencies), whereas ...

show abstract

“…In recent years considerable effort has been put into the investigation of the mechanisms of these enzymes (Ghisla and Massey, 1989;Thorpe and Kim, 1995). Several ofthem have been cloned, expressed, and purified in their recombinant forms (Kieweg et al, 1997;Nandy et al, 1996;Peterson et al, 1995;Mohsen and Vockley, 1995), and the latter have been used extensively for biochemical studies also involving active site directed mutagenesis. While the differences in the primary sequence of recombinant MCADH's compared to those present in mitochondria might be restricted to the first amino acid(s) at the N-terminus, further post-translational changes may occur upon import into mitochondria.…”

Section: Introductionmentioning

confidence: 99%

Medium-Chain Acyl CoA Dehydrogenase: Evidence for Phosphorylation

Macheroux¹,

Sanner²,

Büttner³

et al. 1997

Biological Chemistry

View full text Add to dashboard Cite

Mature medium chain acyl-CoA dehydrogenase isolated from pig kidney (pkMCADH) and originating from mitochondria carries a phosphate group as demonstrated by 31 P-NMR-spectroscopy and chemical analysis. Two broad resonances at -6.3 and -8 ppm are observed and are assigned to the pyrophosphate group of the cofactor FAD. A third, narrow resonance at 4.65 ppm indicates the presence of a phosphomonoester residue. Chemical analysis of intact pkMCADH shows the presence of 3 ± 0.3 phosphates, those of FAD and of an additional covalently attached phosphate. With recombinant, human wild type MCADH expressed in and purified from E. coli only the two FAD phosphates (2 ± 0.35) are found. Similarly, pkMCADH which has been converted to the apoenzyme and reconstituted to holoenzyme also contains 2 ± 0.4 phosphates. The covalently bound phosphate can be hydrolyzed by phosphatase and subsequently removed by dialysis. The phosphate group has no detectable effect on the catalytic activity of the MCADH measured with artificial and natural electron acceptors such as pig electron transferring flavoprotein. However, phosphorylation has a marked effect on protein solubility which is ffi5-fold lower for the dephosphorylated protein.

show abstract

Recombinant Human Liver Medium-Chain Acyl-CoA Dehydrogenase: Purification, Characterization, and the Mechanism of Interactions with Functionally Diverse C8-CoA Molecules

Cited by 27 publications

References 18 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

A Novel Approach to the Characterization of Substrate Specificity in Short/Branched Chain Acyl-CoA Dehydrogenase

Medium-Chain Acyl CoA Dehydrogenase: Evidence for Phosphorylation

Contact Info

Product

Resources

About