Activation, inhibition, and regiospecificity of the lysophospholipase activity of the 85-kDa group IV cytosolic phospholipase A2.
The 85-kDa Group IV calcium-dependent cytosolic phospholipase A2 (cPLA2) catalyzes the hydrolysis of palmitoylglycero-3-phosphocholine to palmitic acid and glycero-3-phosphocholine. Palmitoylglycero-3-phosphocholine exists as a 9:1 equilibrium mixture of the sn-1 and sn-2 isomers, with the fatty acid predominately at the sn-1 position. We have monitored this reaction by 31P NMR to determine which palmitoylglycero-3-phosphocholine isomer is processed by cPLA2. When both lysophospholipid isomers are present in a 1:1 mixture under conditions in which acyl migration is minimized, cPLA2 rapidly consumes both isomers. However, 1-palmitoylglycero-3-phosphocholine is consumed seven times faster than the 2-palmitoylglycero-3-phosphocholine isomer. We have previously reported that this lysophospholipase reaction is accelerated in the presence of glycerol. We now find that this apparent increase in activity is accounted for, in part, by glycerol acting as an alternative acceptor for the cleaved fatty acid, as is the case for this enzyme's phospholipase A2 (PLA2) activity. In contrast, dioleoylglycerol, which accelerates the PLA2 activity, does not act as an acceptor in either the lysophospholipase or the PLA2 reaction, but can affect enzyme activities by altering substrate presentation. We also show that a known inhibitor of the PLA2 activity of cPLA2 is able to inhibit its lysophospholipase activity with a similar IC50 to its PLA2 activity. However, the effect of inhibitors is dependent on the manner in which they are presented to the enzyme.
Loo
R W
RW
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093-0601, USA.
Conde-Frieboes
K
K
Reynolds
L J
LJ
Dennis
E A
EA
D001665:Binding Sites; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D008245:Lysophospholipase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D013237:Stereoisomerism
D004075:Diglycerides; D010741:Phospholipases A; D054467:Phospholipases A2; D008245:Lysophospholipase; D005990:Glycerol