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The N-ethylmaleimide-sensitive cysteine residue in the pH-dependent subunit interactions of malate dehydrogenase
Abstract:
The specific chemical modification by N-ethylmaleimide of a cysteine residue at pH 5.0 in porcine heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) has been shown to result in an enzymatically inactive, monomeric product, which does not reassociate at pH 7.5 to yield the native dimer. In this report, an investigation of proton release and uptake upon NADH binding to the native enzyme and to the N-ethylmaleimide-modified enzyme has implicated the above cysteine residue as being directly linked to the pH-dependent subunit dissociation of mitochondrial malate dehydrogenase. The results are consistent with the view that the modified cysteine residue is not located at the subunit interaction site, although it is probably near this site. A recent study from this laboratory has demonstrated that the monomeric enzyme obtained at pH 5.0 exists in a conformation which is enzymatically inactive and which has an enhanced intrinsic protein fluorescence. Interpretation of protein fluorescence data has suggested that the N-ethylmaleimide modification results in inactivation of the enzyme by preventing the pH-induced conformational change to the active dimer. However, NADH is able to induce reassociation of the N-ethylmaleimide-modified enzyme at pH 7.5 but not at pH 5.0. This reassociation at pH 7.5 is accompanied by a significant regain of enzymatic activity, indicating that NADH binding is able to partially overcome the negative effect of the cysteine modification on the pH-dependent subunit reassociation of mitochondrial malate dehydrogenase.
Insights
Chemical modification of porcine mitochondrial malate dehydrogenase (MDH) inactivates the enzyme. NADH binding partially restores activity by promoting reassociation, suggesting the modified cysteine is near, but not at, the subunit interface.
Area of Science:
- Biochemistry
- Enzymology
- Protein chemistry
Background:
- Porcine heart mitochondrial malate dehydrogenase (MDH) is a dimeric enzyme.
- MDH undergoes pH-dependent subunit dissociation.
- N-ethylmaleimide (NEM) modification of a specific cysteine residue at pH 5.0 yields an inactive monomer.
Purpose of the Study:
- To investigate the role of the cysteine residue modified by NEM in the pH-dependent subunit dissociation of MDH.
- To elucidate the mechanism by which NEM modification affects MDH activity and reassociation.
- To understand the influence of NADH binding on the modified enzyme's conformation and activity.
Main Methods:
- Enzyme kinetics assays to measure activity.
- Proton release/uptake measurements upon NADH binding.
- Protein fluorescence spectroscopy to monitor conformational changes.
- Enzyme reassociation studies at different pH values.
Main Results:
- Proton exchange upon NADH binding implicates the modified cysteine in pH-dependent subunit dissociation.
- NEM modification does not appear to directly block the subunit interaction site.
- NADH binding can induce reassociation and partial activity regain in the NEM-modified enzyme at pH 7.5, but not at pH 5.0.
- The modified cysteine residue is crucial for maintaining the active dimeric conformation.
Conclusions:
- The modified cysteine residue is directly linked to the pH-dependent subunit dissociation of mitochondrial malate dehydrogenase.
- NEM modification prevents the pH-induced conformational change to the active dimer, leading to inactivation.
- NADH binding can partially overcome the inhibitory effects of NEM modification by promoting reassociation and activity recovery.