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Reversible modification of pig heart mitochondrial malate dehydrogenase by pyridoxal 5'-phosphate
Abstract:
1. Pig heart mitochondrial malate dehydrogenase incubated with pyridoxal 5'-phosphate at pH 8.0 and 25 degrees C gradually loses activity. Such inactivation can be largely reversed by dialysis or by addition of L-lysine or L-cysteine, and can be made permanent by NaBH4 reduction. 2. Modification of malate dehydrogenase with pyridoxal 5'-phosphate at 35 degrees C involves two phases, an initial inactivation which is reversible and a slower irreversible second stage. 3. The initial reaction between pyridoxal 5'-phosphate and malate dehydrogenase appears to involve reversible formation of a Schiff base with the epsilon-amino group of a lysine residue. 4. Inactivation of malate dehydrogenase by pyridoxal 5'-phosphate at 10 degrees C involves only the reversible reaction. 5. At 10 degrees C repeated cycles of treatment with pyridoxal 5'-phosphate and NaBH4 reduction lead to a stepwise decline in residual activity. 6. Apparent Km values for malate and NAD+ are unaltered in the partially inactivated enzyme. 7. NAD+ and NADH give only partial protection against pyridoxal 5'-phosphate inactivation. Substrates give no effect.
Insights
Pyridoxal 5'-phosphate inactivates pig heart mitochondrial malate dehydrogenase by modifying lysine residues. This inactivation is reversible initially but can become permanent with NaBH4 reduction, affecting enzyme activity.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Protein modification
Background:
- Mitochondrial malate dehydrogenase is a key enzyme in the citric acid cycle.
- Pyridoxal 5 phosphate is a cofactor that can react with amino groups in proteins.
Purpose of the Study:
- To investigate the mechanism of inactivation of pig heart mitochondrial malate dehydrogenase by pyridoxal 5 phosphate.
- To characterize the reversibility and permanence of the inactivation process.
Main Methods:
- Incubation of purified pig heart mitochondrial malate dehydrogenase with pyridoxal 5 phosphate at various temperatures (10, 25, and 35 degrees C).
- Treatment with sodium borohydride (NaBH4) to assess permanence of modification.
- Dialysis and addition of L-lysine or L-cysteine to evaluate reversibility.
- Determination of kinetic parameters (Km) for malate and NAD+.
Main Results:
- Pyridoxal 5 phosphate causes gradual inactivation of malate dehydrogenase at 25 degrees C, which is reversible by dialysis or L-lysine/L-cysteine but permanent after NaBH4 reduction.
- Inactivation at 35 degrees C proceeds in two phases: an initial reversible phase and a slower irreversible phase, suggesting Schiff base formation with a lysine residue.
- At 10 degrees C, only reversible inactivation occurs. Repeated pyridoxal 5 phosphate/NaBH4 treatment leads to stepwise activity loss. Kinetic parameters (Km) remain unchanged, and substrates offer no protection.
Conclusions:
- Pyridoxal 5 phosphate inactivates malate dehydrogenase primarily through modification of a lysine residue's epsilon-amino group.
- The reaction involves reversible Schiff base formation, which can be stabilized by NaBH4 reduction, leading to irreversible inactivation.
- Enzyme activity loss does not affect substrate binding affinity (Km) for malate or NAD+.