Competitive inhibition of lipolytic enzymes. X. Further delineation of the active site of pancreatic phospholipases A2 from pig, ox and horse by comparing the inhibitory power of a number of (R)-2-acylamino phospholipid analogues.
Two series of (R)-phospholipid analogues, each containing a n-propyl group at the C-1 position and various acylamino functions at the C-2 position have been synthesized and their inhibitory properties towards three mammalian pancreatic phospholipases A2 have been determined. The members of the first series of analogues all contained the zwitter-ionic phosphocholine headgroup which in the second series was replaced by the anionic phosphoglycol function. In the saturated 2-acylamino phospholipids the length of the acyl chain ranged from 8 to 18 carbon atoms. The unsaturated 2-acylamino analogues possessed a chain length of 11 or 18 carbon atoms and contained one, two, three or four double bonds. For inhibitors with a saturated acylamino group, the phospholipases A2 from pig, ox and horse show a sharp optimum in inhibitory power Z for an acyl chain length of 10 carbon atoms. The inhibitory behaviour of the unsaturated acylamino analogues is more complex: both the zwitter-ionic and the anionic inhibitors demonstrate an increase in Z with an increasing number of cis-double bonds but the degree of improvement is dependent on the position of the double bonds. Subsequently the influence of polar groups at carbon position 12 of the dodecanoylamino phospholipids on Z was analyzed. Substitution of the terminal methyl group by an OH-function lowers the inhibitory potency of the three enzymes by a factor of 4 to 5 both in the phosphocholine and phosphoglycol series. Replacement of the methyl group by potentially charged functions (-NH2, -COOH) resulted in a complete loss of inhibitory properties. Blocking of the amino group and carboxyl function by t-butyloxycarbonylation and esterification, respectively, fully restored the inhibitory power. Finally we investigated how changes in the polar headgroup and the presence of aromatic rings at the C-1 or C-2 position influenced the inhibitory potency of the analogues.
Dijkman
R
R
Department of Enzymology and Protein Engineering, C.B.L.E., Utrecht, The Netherlands.
Cox
R
R
van den Berg
L
L
Verheij
H M
HM
De Haas
G H
GH
D000215:Acylation; D000588:Amines / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D006736:Horses; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000494:pharmacology*; D013552:Swine
D000588:Amines; D010743:Phospholipids; D010741:Phospholipases A