Phospholipids chiral at phosphorus. Use of chiral thiophosphatidylcholine to study the metal-binding properties of bee venom phospholipase A2.
It has been shown recently by 31P nuclear magnetic resonance (NMR) that phospholipase A2 (PL A2) from bee venom shows a high degree of stereoselectivity toward the "isomer B" of 1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine (DPPsC) [Bruzik, K., Jiang, R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. We now report a quantitative kinetic study of PL A2 using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and (RP)-, (SP)-, and (RP + SP)-DPPsC by a spectrophotometric assay. The substrates were mixed with Triton X-100 to form mixed micelles, and steady-state kinetic theories were applied. The enzyme was activated by Ca2+, which induced a conformational change of the enzyme, as shown by UV difference spectra. The apparent dissociation constant of Ca2+/PL A2 is 2.5 mM. In the presence of Ca2+, large substrate specificity and stereospecificity in Vmax (in mumol min-1 mg-1) were observed: DPPC, 1850; (RP)-DPPsC, 7.6; (RP + SP)-DPPsC, 64; (SP)-DPPsC, 0.044. On the other hand, relatively small variation in Km was observed, which suggests that the interfacial interaction is relatively nonspecific among the substrates studied. (SP)-DPPsC and Cd2+ were shown as competitive inhibitors for the hydrolysis of DPPC by Ca2+/PL A2. Binding of Cd2+ with apo-PL A2 was also demonstrated by UV difference spectra, with a dissociation constant of 0.59 mM. Activation of apo-PL A2 by Cd2+ was unequivocally demonstrated for (SP)-DPPsC by use of 31P NMR. The Vmax values of Cd2+/PL A2 were DPPC/(RP)-DPPsC/(SP)-DPPsC = 17.6/0.069/0.0044 mumol min-1 mg-1.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001514:Bee Venoms / Q000378:metabolism*; D001667:Binding, Competitive; D002104:Cadmium / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology*; D004789:Enzyme Activation; D007536:Isomerism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010767:Phosphorylcholine; D011663:Pulmonary Surfactants / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D001514:Bee Venoms; D011663:Pulmonary Surfactants; D002104:Cadmium; D010767:Phosphorylcholine; C038151:1,2-dipalmitoyl-sn-glycero-3-thiophosphocholine; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D002122:Calcium Chloride