18O isotope exchange experiments on phospholipase A2 determined by 13C-NMR: monomeric phosphatidylcholine and micellar phosphatidylethanolamine substrates.
Experiments were carried out to determine whether the hydrolytic step or the product release step is the rate-limiting step for non-activated phospholipase A2 hydrolysis (Dennis, E.A. (1983) in The Enzymes, 3rd Edn., Vol 16 (Boyer, P., ed.), pp. 307-353, Academic Press, New York) of mixed micelles of phosphatidylethanolamine and Triton X-100 in the absence of activator phospholipids and of monomeric short chain phosphatidylcholine in the absence of an interface (Lombardo, D. et al. (1986) J. Biol. Chem. 261, 11663-11666). Phospholipase A2-catalyzed exchange of H2(18)O into 1-alkyl-2-[1(13)C]lauroyl-sn-glycero-3-phosphorylethanolamine and into 1-hexanoyl-2-[1-13C]hexanoyl-sn-glycero-3-phosphorylcholine were examined. Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C-NMR. Both the substrate and products of the reactions were examined for 18O incorporation. 18O was incorporated into the fatty acid product, but no incorporation of 18O into the substrate was found. These results suggest that the hydrolytic step is not followed by a higher energy transition state and that it, or a step before it, is rate-limiting. Coupled with kinetic experiments, this strongly suggests that the hydrolytic step is the rate-limiting step. Thus, the role of micellar and membrane interfaces in phospholipase A2 reactions does not appear to be by aiding product removal from the enzyme active site.
Fanni
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Department of Chemistry, University of California, San Diego La Jolla.
Deems
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Dennis
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D002247:Carbon Isotopes; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008956:Models, Chemical; D010103:Oxygen Isotopes; D010713:Phosphatidylcholines / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2
D002247:Carbon Isotopes; D008823:Micelles; D010103:Oxygen Isotopes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2