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Active site mapping studies of malate dehydrogenase : identification of essential amino acid residues by
1Department of Chemistry, Indian Institute of Technology, Kanpur.
Abstract:
Mitochondrial malate dehydrogenase (MDH) was found to be rapidly inactivated by o-phthalaldehyde. MDH-o-phthalaldehyde adduct gives a characteristic absorption maximum at 337 nm. Moreover, this derivative shows fluorescence emission maxima at 405 nm when excited at 337 nm and 280 nm. These results were consistent with isoindole ring formation in which the -SH group of cysteine and epsilon-NH2 group of lysine participate in the reaction. The enzyme was found to be protected against o-phthalaldehyde inactivation by NADH, indicating that the essential residues are present at or near the coenzyme binding site. Stoichiometric results indicate that 4 isoindole derivatives are formed per enzyme molecule upon complete inactivation. However, 90% of the activity loss was accompanied by the formation of 2 moles of isoindole per mole of the enzyme. These approaches give consistent evidence that two cysteines along with two lysines in close proximity are essential for the enzymatic activity.
Insights
Mitochondrial malate dehydrogenase (MDH) is inactivated by o-phthalaldehyde, forming isoindole rings. Essential cysteine and lysine residues near the coenzyme binding site are crucial for MDH enzymatic activity.
Area of Science:
- Biochemistry
- Enzymology
- Mitochondrial function
Background:
- Mitochondrial malate dehydrogenase (MDH) is a key enzyme in the citric acid cycle.
- Understanding the structural and functional roles of active site residues is crucial for enzyme mechanism studies.
Purpose of the Study:
- To investigate the inactivation mechanism of MDH by o-phthalaldehyde.
- To identify essential amino acid residues involved in MDH enzymatic activity.
Main Methods:
- Enzyme inactivation assays using o-phthalaldehyde.
- Spectroscopic analysis (UV-Vis absorption and fluorescence) of MDH-o-phthalaldehyde adducts.
- Determination of reaction stoichiometry.
Main Results:
- O-phthalaldehyde rapidly inactivates MDH through isoindole ring formation involving cysteine and lysine residues.
- NADH protects MDH activity, indicating essential residues are near the coenzyme binding site.
- Stoichiometry suggests two cysteine and two lysine residues are essential for enzymatic activity.
Conclusions:
- The study elucidates the role of specific cysteine and lysine residues in MDH function.
- The findings provide insights into the enzyme's active site structure and coenzyme interaction.
- O-phthalaldehyde serves as a useful probe for studying MDH active site residues.