pH dependence of the reaction rate of p-bromophenacyl bromide and of the binding constants of Ca2+ and an amide-type substrate analog to bovine pancreatic phospholipase A2.
pH dependence of the chemical reaction rates of p-bromophenacyl bromide (BPB) and of the binding constants of Ca2+ to bovine pancreatic active- and pro-phospholipases A2 (PLA2s) was studied at 25 degrees C and ionic strength 0.2. The pH dependence curves of the reaction rates of BPB with both enzymes were biphasic. The amino acid residues participating in the two transitions were ascribed to His 48 and the N-terminal alpha-amino group for the active enzyme and to His 48 and Arg -1 for the proenzyme. The pH dependence curve of Ca2+ binding to the active enzyme was interpreted in terms of participation of Asp 49, His 48, and the alpha-amino group. On the other hand, the curve for the proenzyme was interpreted in terms of participation of Asp 49, His 48, and Arg -1. The Ca2+ and pH dependence of the binding constant of a potent competitive inhibitor, monodispersed (R)-2-dodecanoylamino-1-hexanol-phosphocholine (amide-PC), to bovine pancreatic active-PLA2 was also studied. The binding of amide-PC was markedly facilitated by Ca2+ binding to the enzyme, whereas that of a genuine substrate, monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), was independent of Ca2+ binding. The pH dependence curve of the binding constant of the amide-PC showed one transition, and this was interpreted in terms of participation of His 48, whereas the binding of the diC6PC was independent of the ionization state of His 48. The difference in the Ca2+ dependence for the bindings of the diC6PC and amide-PC was considered to arise from the fact that the amide group of amide-PC can form a hydrogen bond with His 48, whereas the genuine substrate cannot form such a hydrogen bond.
Fujii
S
S
Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Japan.
Meida
M
M
Tani
T
T
Inoue
S
S
Iwama
S
S
Katsumura
S
S
Ikeda
K
K
D000098:Acetophenones / Q000378:metabolism*; D000577:Amides / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity
C092445:1,2-dihexanoyl-sn-glycero-3-phosphorylcholine; D000098:Acetophenones; D000577:Amides; D009943:Organophosphorus Compounds; D010713:Phosphatidylcholines; C093914:2-dodecanoylamino-1-hexanolphosphocholine; D010741:Phospholipases A; D054467:Phospholipases A2; C006821:4-bromophenacyl bromide; D002118:Calcium