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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.

Abstract：

: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.

journal_name

Arch Biochem Biophys

journal_title

Archives of biochemistry and biophysics

authors

Fujii S,Meida M,Tani T,Inoue S,Iwama S,Katsumura S,Ikeda K

doi

10.1006/abbi.1998.0681

subject

Has Abstract

pub_date

1998-06-01 00:00:00

pages

73-82

issue

eissn

0003-9861

issn

1096-0384

pii

S0003-9861(98)90681-6

journal_volume

354

pub_type

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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.