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Alanine dehydrogenase from Enterobacter aerogenes: purification, characterization, and primary structure
E K Chowdhury1, T Saitoh, S Nagata
1Department of Bioresources Science, Kochi University, Japan.
Bioscience, Biotechnology, and Biochemistry
|February 11, 1999
Summary
Enterobacter aerogenes ICR 0220 alanine dehydrogenase was purified and characterized. This enzyme exhibits unique kinetic properties and structural differences, including the absence of cysteine residues, setting it apart from other alanine dehydrogenases.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Alanine dehydrogenase (EC 1.4.1.1) plays a crucial role in amino acid metabolism.
- Understanding enzyme kinetics and structure is vital for biochemical research.
- Enterobacter aerogenes is a model organism for studying bacterial enzymes.
Purpose of the Study:
- To purify and characterize alanine dehydrogenase from Enterobacter aerogenes ICR 0220.
- To elucidate the enzyme's kinetic properties, coenzyme requirements, and reaction mechanism.
- To compare the enzyme's characteristics with other known alanine dehydrogenases and analyze its gene sequence.
Main Methods:
- Enzyme purification using standard biochemical techniques.
- Enzyme activity assays at varying pH and substrate concentrations.
- Kinetic studies including initial-velocity and product inhibition experiments.
- Coenzyme analog analysis.
- Gene cloning into Escherichia coli JM109 and nucleotide sequencing.
- Bioinformatic analysis of the deduced amino acid sequence.
Main Results:
- Alanine dehydrogenase was purified to homogeneity, revealing a molecular mass of 245 kDa composed of six identical subunits.
- The enzyme displayed optimal activity at pH 10.9 for deamination and pH 8.7 for amination, requiring NAD+ as a coenzyme.
- Kinetic studies indicated a sequential ordered mechanism for L-alanine deamination, with specific Michaelis constants (Km) determined for substrates and products.
- The Km for L-alanine was notably low compared to other studied alanine dehydrogenases.
- Gene sequencing revealed high similarity to Bacillus subtilis alanine dehydrogenase, but the Enterobacter enzyme lacks cysteine residues.
Conclusions:
- The purified alanine dehydrogenase from Enterobacter aerogenes possesses distinct kinetic and structural features.
- The enzyme's unique characteristics, particularly the absence of cysteine, differentiate it from other known alanine dehydrogenases.
- The study provides valuable insights into the diversity and evolution of alanine dehydrogenases.