Cytosolic phospholipase A2 shows burst kinetics consistent with the slow, reversible formation of a dead-end complex.
Cytosolic phospholipase A2 catalyzes the hydrolysis of the sn-2 ester of arachidonate-containing phospholipids. In the present research, a "burst" of arachidonate which precedes a somewhat slower, linear rate (upsilon) of product formation was observed and characterized using covesicles of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) containing <10 mol% 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate. The magnitude of the burst (pi) was enzyme dependent, in both the presence and absence of glycerol. Upon subsequent addition of enzyme after the primary burst was complete, a second burst of arachidonate production was observed. This is consistent with the effect resulting from an enzyme effect and not from changes in the substrate. The use of 1,2-dioleoyl-sn-glycero-3-phosphomethanol as the carrier phospholipid instead of DMPM greatly reduced the rate of hydrolysis without a large effect on the pi/upsilon ratio, consistent with the burst not being the result of limitations in the lateral diffusion rate of phospholipids within the covesicles. When the assay is performed in the presence of glycerol, the burst phenomenon was also observed with the monoarachidonoyl glycerol transacylase product which shows that the effect occurs through a common mechanism. The burst and subsequent linear rate of hydrolysis are highly temperature dependent, with a pronounced increase in the pi/upsilon ratio as the temperature is increased from 35 to 45 degrees C. A mechanism in which a slow equilibrium between an active and less active (inactive) state of substrate-bound enzyme is proposed. This may provide a means by which the enzyme is switched off after a few hundred turnovers in order to prevent unabated phospholipid hydrolysis in cells which may be deleterious to membrane integrity.
Guenther
Matthew G
MG
Drug Discovery and Exploratory Development, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA.
Witmer
Mark R
MR
Burke
James R
JR
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002384:Catalysis; D003600:Cytosol / Q000201:enzymology; D005989:Glycerides / Q000378:metabolism; D006801:Humans; D007313:Insecta / Q000166:cytology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D013696:Temperature
D005989:Glycerides; D010743:Phospholipids; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2