Purification and characterization of an allosteric fructose-1,6-bisphosphate aldolase from germinating mung beans (Vigna radiata).
Cytosolic fructose-1,6-P(2) (FBP) aldolase (ALD(c)) from germinated mung beans has been purified 1078-fold to electrophoretic homogeneity and a final specific activity of 15.1 micromol FBP cleaved/min per mg of protein. SDS-PAGE of the final preparation revealed a single protein-staining band of 40 kDa that cross-reacted strongly with rabbit anti-(carrot ALD(c))-IgG. The enzyme's native M(r) was determined by gel filtration chromatography to be 160 kDa, indicating a homotetrameric quaternary structure. This ALD is a class I ALD, since EDTA or Mg(2+) had no effect on its activity, and was relatively heat-stable losing 0-25% of its activity when incubated for 5 min at 55-65 degrees C. It demonstrated: (i) a temperature coefficient (Q(10)) of 1.7; (ii) an activation energy of 9.2 kcal/mol active site; and (iii) a broad pH-activity optima of 7.5. Mung bean ALD(c) is bifunctional for FBP and sedoheptulose-1,7-P(2) (K(m) approximately 17 microM for both substrates). ATP, ADP, AMP and ribose-5-P exerted inhibitory effects on the activity of the purified enzyme. Ribose-5-P, ADP and AMP functioned as competitive inhibitors (K(i) values=2.2, 3.1 and 7.5mM, respectively). By contrast, the addition of 2mM ATP: (i) reduced V(max) by about 2-fold, (ii) increased K(m)(FBP) by about 4-fold, and (iii) shifted the FBP saturation kinetic plot from hyperbolic to sigmoidal (h=1.0 and 2.6 in the absence and presence of 2mM ATP, respectively). Potent feedback inhibition of ALD(c) by ATP is suggested to help balance cellular ATP demands with the control of cytosolic glycolysis and respiration in germinating mung beans.
Lal
Ashish
A
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi-221 005, India.
Plaxton
William C
WC
Kayastha
Arvind M
AM
D000494:Allosteric Regulation; D002412:Cations; D007887:Fabaceae / Q000201:enzymology*; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D018525:Germination; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
D002412:Cations; D005634:Fructose-Bisphosphate Aldolase