Alpha-amylase from mung beans (Vigna radiata)--correlation of biochemical properties and tertiary structure by homology modelling

Phytochemistry. 2007 Jun;68(12):1623-31. doi: 10.1016/j.phytochem.2007.04.006. Epub 2007 May 23.

Abstract

Alpha-amylase from germinated mung beans (Vigna radiata) has been purified 600-fold to electrophoretic homogeneity and a final specific activity of 437 U/mg. SDS-PAGE of the final preparation revealed a single protein band of 46 kDa. The optimum pH was 5.6. The energy of activation was determined to be 7.03 kcal/mol in the temperature range 15-55 degrees C. Km for starch was 1.6 mg/mL in 50 mM sodium acetate buffer, pH 5.5. Thermal inactivation studies at 70 degrees C showed first-order kinetics with rate constant (k) equal to 0.005 min(-1). Mung bean alpha-amylase showed high specificity for its primary substrate starch. Addition of EDTA (10 mM) caused irreversible loss of activity. Mung bean alpha-amylase is inhibited in a non-competitive manner by heavy metal ions, for example, mercury with a Ki of 110 microM. Homology modelling studies with mung bean alpha-amylase using barley alpha-amylases Amy 1 and Amy 2 as templates showed a very similar structure as expected from the high sequence identity. The model showed that alpha-amylase from mung beans has no sugar-binding site, instead it has a methionine. Furthermore, instead of two tryptophans, it has Val(277) and Lys(278), which are the conserved residues, important for proper folding and conformational stability.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Edetic Acid / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Fabaceae / enzymology*
  • Hordeum / enzymology
  • Hydrogen-Ion Concentration
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Plant Proteins / chemistry*
  • Plant Proteins / isolation & purification
  • Protein Structure, Tertiary
  • Sequence Alignment
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Structural Homology, Protein
  • Substrate Specificity
  • Temperature
  • alpha-Amylases / chemistry*
  • alpha-Amylases / isolation & purification

Substances

  • Plant Proteins
  • Edetic Acid
  • alpha-Amylases