J. Biochem, 1983, Vol. 94, No. 1 223-232
© 1983 Japanese Biochemical Society
research-article |
Bindings of Cobra Venom Phospholipases A2 to Micelles of n-Hexadecylphosphorylcholine1
*Department of Biology, Faculty of Science, Osaka University Toyonaka, Osaka 560
**Kyoto University Sakyo-ku, Kyoto, Kyoto 606
3To whom correspondence should be sent.
Bindings of cobra venom phospholipases A2 to micelles of n-hexadecylphosphoryl-choline were studied by the tryptophyl fluorescence method 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 these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of substrate (monomer) molecules, N=10, 5 or 13 for N. naja atra apoenzyme and its Ca2+ complex, and N. naja kaouthia apoenzyme, respectively. The binding constant of the enzymes to the micelle, Kmic = 0.18–3.1 × 108 M–1, was 9–160 times greater than that to the monomeric substrate, Kmon = 2×104 M–1 (Teshima et al. (1981) J. Biochem. 89, 1163–1174). This was interpreted in terms of the presence of an additional substrate-binding site in the enzyme molecule. The binding constant of the enzyme-Ca2+ complex to the micelle was smaller than that for the apoenzyme over a wide range of pH.
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 5.4 to 5.53 and 7.55 to 7.95. The pH dependence curve for the Ca2+ complex, which lacked the former transition, was interpreted in terms of the pK shift of only a single ionizable group from 7.25 to 7.55. The former ionizable group was assigned as Asp 49, to which Ca2+ can coordinate, and the latter as His 48 in the active site on the basis of the reported pK values of these ionizable groups in the apoenzyme and Ca2+ complex (Teshima et al. (1981) J. Biochem. 89, 13–20 and Teshima et al. (1982) J. Biochem. 91, 1777–1788). No participation of the 
-amino group with a pK value of 8.55 was observed.
1 This work was supported in part by a grant for scientific research from the Ministry of Education, Science, and Culture of Japan. A part of this work was presented at the 33rd Symposium on Protein Structure held at Osaka in October, 1982.
2 Present address: Hoshi Institute of Pharmaceutical Sciences, Shinagawa-ku, Tokyo, Tokyo 142.
4 Present address: Department of Biology, Gifu College of Pharmacy, Mitahora-Higashi, Gifu 502.