Dimerization and activation of porcine pancreatic phospholipase A2 via substrate level acylation of lysine 56.
The porcine pancreatic phospholipase A2-catalyzed hydrolysis of the water-soluble chromogenic substrate 4-nitro-3-octanoyloxybenzoate shows an initial latency phase similar to the one observed in the hydrolysis of aggregated phospholipids by the same enzyme. We report here that during the latency phase the enzyme undergoes a slow, autocatalytic, substrate-level acylation whereby in a few of the catalytic events the scissile octanoyl group of the substrate, normally transferred to water, is transferred to the epsilon-amino group of lysine 56. The N epsilon 56-octanoylphospholipase shows a strong tendency to dimerize in solution and thus may be separated from the monomeric native enzyme by gel filtration. Octanoylation of Lys-56 activates the enzyme some 180-fold toward 4-nitro-3-octanoyloxybenzoate and more than 100-fold toward monolayers of 1,2-didecanoyl-sn-glycero-3-phosphocholine. Acylation also attends the enzymatic hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine with the incorporation of 1 eq of palmitate. Kinetic analysis of the early phase of reaction with 4-nitro-3-octanoyloxybenzoate shows that in this initial step the rate of activation is first order with respect to enzyme and substrate. A much more rapid, autocatalytic activation occurs in the later phases of the reaction where the activation of the enzyme is catalyzed by the activated enzyme itself. These findings with porcine pancreatic phospholipase A2, together with those relative to a snake venom enzyme monomer (Cho, W., Tomasselli, A. G., Heinrikson, R. L., and Kézdy, F. J. (1988) J. Biol. Chem. 263, 11237-11241), strongly support the proposal that interfacial activation of monomeric phospholipases is due to substrate-level autoacylation resulting in fully potentiated dimeric enzymes.
Tomasselli
A G
AG
Biopolymer Chemistry Unit, Upjohn Company, Kalamazoo, Michigan 49001.
Hui
J
J
Fisher
J
J
Zürcher-Neely
H
H
Reardon
I M
IM
Oriaku
E
E
Kézdy
F J
FJ
Heinrikson
R L
RL
D000215:Acylation; D000818:Animals; D004789:Enzyme Activation; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D013552:Swine
D046911:Macromolecular Substances; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D008239:Lysine