pH dependence of the binding constant of a phospholipase A2 from Agkistrodon halys blomhoffii venom to micelles of n-hexadecylphosphorylcholine.
The phospholipase A2 from the venom of A. halys blomhoffii was titrated with micellar n-hexadecylphosphorylcholine (an analog of lysolecithin) by following the tryptophyl fluorescence change at 25 degrees C and ionic strength 0.1. The data were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 10.0 and 6.7 for the apoenzyme and its Ca2+ complex, respectively. The binding constant of the enzyme to the substrate micelle was found to be enhanced by Ca2+ binding to the enzyme. The pH dependence of the binding constant of the apoenzyme to the micelle was well interpreted in terms of pK shifts of two ionizable groups from 5.16 to 5.67 and from 6.45 to 6.6. The pH-dependence curve for the enzyme-Ca2+ complex, which lacked the former transition, was interpreted in terms of the pK shift of a single ionizable group from 5.55 to 5.76. The former ionizable group was assigned as Asp 49, to which Ca2+ ion can coordinate, and the latter as His 48 in the active site. No participation of the alpha-amino group with a pK value of 7.30 was observed. The binding constant of the enzyme to the substrate micelle, Kmic = 0.45-2.3 X 10(6) M-1, was found to be far greater than that to the monomeric substrate, Kmon = 0.2-1.0 X 10(4) M-1. This was interpreted in terms of the presence of an additional weak substrate-binding site in the enzyme molecule.
D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002794:Choline / Q000031:analogs & derivatives*; D003435:Crotalid Venoms / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008433:Mathematics; D008823:Micelles; D008956:Models, Chemical; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
C031442:Agkistrodon venoms; D003435:Crotalid Venoms; D008823:Micelles; D010767:Phosphorylcholine; C039128:miltefosine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002794:Choline