Kinetic analysis of a high molecular weight phospholipase A2 from rat kidney: divalent metal-dependent trapping of enzyme on product-containing vesicles.
The kinetics of hydrolysis of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine vesicles catalyzed by the high molecular weight phospholipase A2 from rat kidney show an anomalous behavior. The reaction progress lasts for several minutes and then stops after only 5-10% of the available substrate has been hydrolyzed. Addition of more enzyme but not more substrate leads to a new round of hydrolysis. Although this initially suggested that the enzyme becomes inactivated during the turnover, such a conclusion could not be substantiated. Addition of buffer containing 0.15 M NaCl and bovine serum albumin to the reaction after the progress ceased leads to the re-initiation of the lipolysis. The enzyme is not strongly inhibited by the reaction products. Although the enzyme does not bind irreversibly to vesicles composed of pure 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine, it does become irreversibly trapped on vesicles that contain a critical mole percentage of reaction products. This trapping is the most likely explanation for the cessation of the reaction progress. Both the binding of enzyme to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine vesicles and the hydrolysis of 1-stearoyl-2-[3H]arachidonyl-sn-glycerophosphocholine contained in these vesicles require the presence of products. Furthermore, the trapping of enzyme is independent of catalytic turnover. The trapping is sensitive to the structure of the fatty acid present in the vesicles and requires the presence of divalent metals (either Ca2+, Sr2+, Ba2+, or Mg2+). Since the concentrations of the metals needed for the enzymatic activity correlate with the amounts needed to promote the trapping, it is suggested that the role of the metal is only to promote the interfacial binding of the enzyme.
Ghomashchi
F
F
Department of Chemistry, University of Washington, Seattle 98195.
Schüttel
S
S
Jain
M K
MK
Gelb
M H
MH
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D008970:Molecular Weight; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010742:Phospholipid Ethers / Q000378:metabolism; D014280:Triglycerides / Q000378:metabolism
D002413:Cations, Divalent; D008670:Metals; D010742:Phospholipid Ethers; D014280:Triglycerides; D015060:1,2-Dipalmitoylphosphatidylcholine; C043051:1-O-hexadecyl-2-arachidonyl-sn-glycero-3-phosphocholine; D010741:Phospholipases A; D054467:Phospholipases A2