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L-Malate dehydrogenase from Pseudomonas stutzeri: purification and characterization
1Department of Agricultural Biotechnology, The Agricultural University of Athens, Greece.
Archives of Biochemistry and Biophysics
|January 1, 1997
Summary
Purified Pseudomonas stutzeri L-malate dehydrogenase (MDH) exhibits properties similar to eukaryotic mitochondrial MDH. This bacterial enzyme
Area of Science:
- Biochemistry
- Enzymology
- Protein Purification
Background:
- L-Malate dehydrogenase (MDH) is a crucial enzyme in cellular metabolism.
- Understanding the structural and functional properties of bacterial MDH provides insights into enzyme evolution.
Purpose of the Study:
- To purify and characterize L-malate dehydrogenase (MDH) from Pseudomonas stutzeri.
- To investigate the enzyme's kinetic mechanism, substrate interactions, and inactivation by a specific dye.
- To compare the properties of bacterial MDH with its eukaryotic counterparts.
Main Methods:
- Two-step purification: anion-exchange and affinity chromatography.
- Enzyme kinetics: initial velocity, product inhibition, and binding studies.
- Chemical modification: inactivation with Vilmafix Blue A-R (VBAR) dye.
- Spectroscopic analysis: sulfhydryl group determination.
Main Results:
- Purified P. stutzeri MDH has a molecular mass of 66,500 Da, composed of two identical subunits.
- The enzyme follows an ordered Bi-Bi mechanism and forms a ternary complex.
- Inactivation by VBAR dye involves noncovalent complex formation (Kd 84.6 µM) followed by covalent modification.
- One sulfhydryl group per subunit is located in the coenzyme binding site.
- P. stutzeri MDH shares significant similarities with mitochondrial eukaryotic MDH.
Conclusions:
- The purification and characterization of P. stutzeri MDH reveal its ordered Bi-Bi mechanism and susceptibility to dye inactivation.
- The structural and catalytic similarities suggest a potential evolutionary link between bacterial and mitochondrial MDH.
- This study supports the hypothesis that mitochondrial MDH may have evolved prior to cytoplasmic MDH.