title: Presence of glycerol masks the effects of phosphorylation on the catalytic efficiency of cytosolic phospholipase A2.
abstract: Cytosolic phospholipase A2 catalyzes the selective release of arachidonic acid from the sn-2 position of phospholipids and is believed to play a key cellular role in the generation of arachidonic acid. The enzymatic activity of cPLA2 is affected by several mechanisms, including substrate presentation and the phosphorylation state of the enzyme. Using covesicles of 1-palmitoy1-2-arachidonoyl-[arachidonoyl-1-14C]-8n-glycero-3 -phosphocholine and 1,2-dimyristoyl-phosphatidylmethanol as substrate, the effects of phosphorylation on the interfacial binding and catalytic constants were investigated. Phosphorylated and dephosphorylated enzyme forms were shown to have identical values of 2.6 microM for KMapp, an equilibrium dissociation constant which consists of the intrinsic dissociation constant from the lipid/water interface (Ks) and the dissociation constant for phospholipid from the active site (KM*). Moreover, the values of KM* for phosphorylated and dephosphorylated enzyme did not differ significantly (0.4 +/- 0.1 and 0.2 +/- 0.1, respectively). However, dephosphorylation of the enzyme reduced the value of kcat by 39%. The phosphorylation state of the enzyme had no effect on either the cooperativity shown by this enzyme or the thermal stability of the enzyme. Surprisingly, the presence of glycerol (4 M) masks the effect of phosphorylation on kcat. Instead, glycerol increased the value of kcat by 440% for the phosphorylated enzyme and by 760% for the dephosphorylated form. Moreover, addition of glycerol had only small effects on KMapp. the increase in the kcat upon addition of glycerol results from a substantial decrease in the activation energy from 29.4 to 14.8 kcal. mol-1. To determine whether the effects of phosphorylation of the enzyme or addition of glycerol are unique to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With these membranes, the dephosphorylated enzyme was only 21% less active than the phosphorylated enzyme. In the presence of glycerol, there was no detectable difference the two enzyme forms, and the rate of hydrolysis was increased by 300-390% over that measured in the absence of glycerol. These results suggest that the catalytic efficiency of the phosphorylated enzyme is not particularly relevant to its activation in vivo. Moreover, it may be that glycerol is mimicking the effect of some unidentified factor which greatly enhances the catalytic efficiency of the enzyme.
authors.affiliation: Department of Dermatology Discovery Research, Bristol-Myers Squibb Pharmaceutical Research Institute, Buffalo, New York 14213, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Burke
authors.forename: M G
authors.initials: MG
authors.lastname: Guenther
authors.forename: M R
authors.initials: MR
authors.lastname: Witmer
authors.forename: J A
authors.initials: JA
authors.lastname: Tredup
authors.forename: M E
authors.initials: ME
authors.lastname: Hail
authors.forename: R
authors.initials: R
authors.lastname: Micanovic
authors.forename: J J
authors.initials: JJ
authors.lastname: Villafranca
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005989:Glycerides / Q000378:metabolism; D005990:Glycerol / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D013058:Mass Spectrometry; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000494:pharmacology; D010766:Phosphorylation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D014407:Tumor Cells, Cultured
chemical_list: D005989:Glycerides; D008081:Liposomes; D010743:Phospholipids; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D000135:Acid Phosphatase; D005990:Glycerol; D002118:Calcium