Biochemical characterization of an aspartic protease from Vigna radiata: Kinetic interactions with the classical inhibitor pepstatin implicating a tight binding mechanism.
Aspartic proteases are the focus of recent research interest in understanding the physiological importance of this class of enzymes in plants. This is the first report of an aspartic protease from the seeds of Vigna radiata. The aspartic protease was purified to homogeneity by fractional ammonium sulfate precipitation and pepstatin-A agarose affinity column. It was found to have a molecular weight of 67,406 Da by gel filtration chromatography. SDS-PAGE analysis revealed the presence of a heterodimer with subunits of molecular weights of 44,024 and 23,349 Da respectively. The enzyme was pH stable with the amino acid analysis confirming the molecular weight of the protein. The substrate cleavage site as analyzed by using the synthetic substrate was found to be the Phe-Tyr bond. The kinetic interactions of the enzyme were studied with the universal inhibitor, pepstatin A. This is the first report on the interactions of a plant aspartic protease with pepstatin-A, an inhibitor from a microbial source. A competitive one-step mechanism of binding is observed. The progress curves are time-dependent and consistent with tight binding inhibition. The K(i) value of the reversible complex of pepstatin with the enzyme was 0.87 microM whereas the overall inhibition constant K(i)* was 0.727 microM.
Kulkarni
Aarohi
A
Rao
Mala
M
D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004795:Enzyme Stability; D007887:Fabaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010436:Pepstatins / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D013379:Substrate Specificity; D013696:Temperature
D004791:Enzyme Inhibitors; D010436:Pepstatins; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin