Calcium and magnesium dependence of phospholipase A2-catalyzed hydrolysis of phosphatidylcholine small unilamellar vesicles.
The Ca2+ requirement for lipid hydrolysis catalyzed by phospholipase A2 from Agkistrodon piscivorus piscivorus (App-D49) and porcine pancreas has been examined using small, unilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC SUV). Hydrolysis was affected by product inhibition even at early times, and the extent of this inhibition depended on the concentration of divalent cations. The Ca2+ requirement for half-maximal rates of hydrolysis reflected, in part, this non-catalytic role of divalent cations. The presence of 10 mM Mg2+, a cation which does not support catalysis, reduced the Ca2+ required for half-maximal rates of hydrolysis from millimolar concentrations to 40 microM for App-D49. Since the dissociation constant of the enzyme for Ca2+ in solution is 2 mM, these results indicate a change in the interaction of the enzyme with Ca2+ under catalytic conditions. The kinetic dissociation constant of Ca2+ for the pancreatic enzyme was 20 microM which is substantially lower than the dissociation constant in solution, 0.35 mM. The similarity of apparent kinetic dissociation constants for these enzymes suggests that structurally similar features determine the affinity for Ca2+ under catalytic conditions. Evidence is presented that the affinity of phospholipase A2 for Ca2+ changes subsequent to the initial interaction of the enzyme with the substrate interface. However, the apparent Michaelis constant, KMapp, for App-D49, 0.03-0.06 mM, is independent of [Ca2+] and is about the same as the equilibrium dissociation constant for DPPC SUV, 0.14 mM. We thus suggest that KMapp is a steady-state constant.
Lathrop
B K
BK
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Biltonen
R L
RL
D015060:1,2-Dipalmitoylphosphatidylcholine; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D003435:Crotalid Venoms / Q000201:enzymology; D007700:Kinetics; D008066:Lipolysis; D008274:Magnesium / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology; D010713:Phosphatidylcholines / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D013552:Swine
D003435:Crotalid Venoms; D010713:Phosphatidylcholines; D015060:1,2-Dipalmitoylphosphatidylcholine; D010741:Phospholipases A; D054467:Phospholipases A2; D008274:Magnesium; D002118:Calcium