An essential methionine in pig kidney general acyl-CoA dehydrogenase

Mizzer, John P.; Thorpe, Colin

doi:10.1021/bi00565a006

Cited by 10 publications

(9 citation statements)

References 18 publications

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“…As reported by Mizzer & Thorpe (1980) for the pig kidney enzyme, the thiol groups of pig liver GAD reacted sluggishly with thiol-blocking reagents. When GAD was treated with a 12-fold excess of 4,4'dithiodipyridine, only 0.66 thiol group/enzyme FAD group had reacted in 80 min, whereas 7.3 thiol groups/enzyme FAD group reacted in less than 2 min in the presence of 4 M-guanidinium chloride, in close agreement with the total number of half-cystine residues/enzyme FAD group determined by amino acid analysis of the pig kidney enzyme (Thorpe et al, 1979).…”

Section: Effect Of Modification Of Lysine Residues In Gad and Etf On supporting

confidence: 54%

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase

Steenkamp¹

1987

Biochemical Journal

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The interaction between pig liver mitochondrial electron-transfer flavoprotein (ETF) and general acyl-CoA dehydrogenase (GAD) was investigated by means of the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Neither ETF or GAD contained reactive thiol groups. The substitution of 9.4 lysine residues/FAD group in GAD with pyridyl disulphide structures did not affect the catalytic activity of the enzyme. Thiol groups were introduced into ETF by thiolation with methyl 4-mercaptobutyrimidate. ETF containing 10.5 reactive thiol groups/FAD group showed undiminished electron-acceptor activity with respect to GAD. The reaction of thiolated ETF and GAD containing pyridyl disulphide structures resulted in a decreased staining intensity of the small subunit of ETF on SDS/polyacrylamide-gel electrophoresis. Preferential cross-linking of the smaller subunit of ETF to GAD did not take place when ETF was first treated with SDS, but was unaffected by reduction of GAD by octanoyl-CoA.

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Section: Effect Of Modification Of Lysine Residues In Gad and Etf On supporting

confidence: 54%

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase

Steenkamp¹

1987

Biochemical Journal

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“…Alkylating reagents including iodoacetate, methyl iodide, iodoacetamide, and iodoacetic acid, all of which react with Met in a pH range of 2–4. Furthermore, there are only a few demonstrations where these reactions occur under gentle conditions (iodoacetate with Met residues in pig kidney general acyl-CoA dehydrogenase pH = 6.6) . Thus, the likely disruption of protein native structure by placing it in an acidic environment make this reagent too risky as a footprinter.…”

Section: Targeted-labeling Reagentsmentioning

confidence: 99%

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

2020

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Proteins adopt different higher-order structures (HOS) to enable their unique biological functions. Understanding the complexities of protein higher-order structures and dynamics requires integrated approaches, where mass spectrometry (MS) is now positioned to play a key role. One of those approaches is protein footprinting. Although the initial demonstration of footprinting was for the HOS determination of protein/nucleic acid binding, the concept was later adapted to MS-based protein HOS analysis, through which different covalent labeling approaches “mark” the solvent accessible surface area (SASA) of proteins to reflect protein HOS. Hydrogen–deuterium exchange (HDX), where deuterium in D2O replaces hydrogen of the backbone amides, is the most common example of footprinting. Its advantage is that the footprint reflects SASA and hydrogen bonding, whereas one drawback is the labeling is reversible. Another example of footprinting is slow irreversible labeling of functional groups on amino acid side chains by targeted reagents with high specificity, probing structural changes at selected sites. A third footprinting approach is by reactions with fast, irreversible labeling species that are highly reactive and footprint broadly several amino acid residue side chains on the time scale of submilliseconds. All of these covalent labeling approaches combine to constitute a problem-solving toolbox that enables mass spectrometry as a valuable tool for HOS elucidation. As there has been a growing need for MS-based protein footprinting in both academia and industry owing to its high throughput capability, prompt availability, and high spatial resolution, we present a summary of the history, descriptions, principles, mechanisms, and applications of these covalent labeling approaches. Moreover, their applications are highlighted according to the biological questions they can answer. This review is intended as a tutorial for MS-based protein HOS elucidation and as a reference for investigators seeking a MS-based tool to address structural questions in protein science.

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“…Unless otherwise stated, all buffers contained 0.3 mM EDTA. Concentrations of native, carboxymethylated, and 8-Cl-FAD-substituted acyl-CoA dehydrogenase were determined spectrophotometrically using the following molar extinction coefficients: 15.4 mM-1 cm-1 at 446 nm (Thorpe et al, 1979); 15.3 mM-1 cm-1 at 445 nm (Mizzer & Thorpe, 1980); and 14.0 mM'1 cm-1 at 440 nm (Thorpe & Massey, 1983), respectively. Anaerobic titrations were performed as in Gorelick et al (1982).…”

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confidence: 99%

“…Preparation of Carboxymethylated Acyl-CoA Dehydrogenase. Carboxymethylation with 30 mM iodoacetic acid was performed as described previously (Mizzer & Thorpe, 1980), except excess reagents were removed by centrifuge ultrafiltration (Centricon PM 10 microconcentrators), washing with 100 mM phosphate buffers, pH 6.6. The modified acyl-CoA dehydrogenase exhibited 5-6% of the activity shown by the native enzyme in the phenazine methosulfate/2,6-dichlorophenolindophenol assay (Thorpe, 1981) and in the ferricenium assay (Lehman et al, 1990) using 30 µ octanoyl-CoA.…”

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confidence: 99%

Reductive half-reaction in medium-chain acyl-CoA dehydrogenase: modulation of internal equilibrium by carboxymethylation of a specific methionine residue

Cummings¹,

Lau²,

Powell³

et al. 1992

Biochemistry

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Pig kidney medium-chain acyl-CoA dehydrogenase is specifically alkylated at a methionine residue by treatment with iodoacetate at pH 6.6. This residue corresponds to Met249 in the human medium-chain acyl-CoA dehydrogenase sequence [Kelly, D. P., Kim, J. J., Billadello, J. J., Hainline, B. E., Chu, T. W., & Strauss, A. W. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 4068-4072]. The S-carboxymethylated dehydrogenase shows a drastically lowered affinity for octanoyl-CoA (from submicromolar to 65 microM), but retains about 23% of the maximal activity of the native enzyme. In addition, alkylation perturbs the internal redox equilibrium: E.FADox.octanoyl-CoA K2 in equilibrium with E.FAD2e.octenoyl-CoA K2 ranges from about 9 for the native enzyme to about 0.2 for the homogeneously modified protein. This effect is not due to a significant change in the redox potential of the free enzyme upon alkylation. Rather, carboxymethylation weakens the preferential binding of enoyl-CoA product to the reduced enzyme (K3) compared to octanoyl-CoA binding to the oxidized dehydrogenase (K1) that is required to pull the substrate thermodynamically uphill. Thus, the ratio of dissociation constants, K1/K3, decreases from about 15,000 for the native enzyme to only 330 upon carboxymethylation of Met249. Binding studies with a variety of acyl-CoA analogues and manipulation of enzyme redox potentials by substitution of the natural prosthetic group by 8-Cl-FAD confirm the thermodynamic effects of alkylation.

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An essential methionine in pig kidney general acyl-CoA dehydrogenase

Cited by 10 publications

References 18 publications

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase

Preferential cross-linking of the small subunit of the electron-transfer flavoprotein to general acyl-CoA dehydrogenase

Mass Spectrometry-Based Protein Footprinting for Higher-Order Structure Analysis: Fundamentals and Applications

Reductive half-reaction in medium-chain acyl-CoA dehydrogenase: modulation of internal equilibrium by carboxymethylation of a specific methionine residue

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