J. Biochem, 1984, Vol. 96, No. 5 1427-1436
© 1984 Japanese Biochemical Society
research-article |
pH Dependence of the Binding Constant of a Phospholipase A2 from Agkistrodon halys blomhoffii Venom to Micelles of n-Hexadecylphosphorylcholine1
*Department of Biology, Faculty of Science, Osaka University Toyonaka, Osaka 560
**Hoshi Institute of Pharmaceutical Sciences Shinagawa-ku, Tokyo 142
2 To whom correspondence should be sent. present address: Department of Biochemistry, Osaka College of Pharmacy, 2-10-65 Kawai, Matsubara, Osaka 580.
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°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 
-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 106 M–1, was found to be far greater than that to the monomeric substrate, Kmon= 0.2–1.0x 104 M–1. This was interpreted in terms of the presence of an additional weak substrate-binding site in the enzyme molecule.
1 This work was supported in part by a Grant-in-Aid for Scientific Research (No. 57380015) from the Ministry of Education, Science and Culture of Japan. A part of this work was presented at the 56th Congress of the Japanese Biochemical Society held at Fukuoka in September, 1983.
3Present address: Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka 565.