Role of 57-72 loop in the allosteric action of bile salts on pancreatic IB phospholipase A2: Regulation of fat and cholesterol homeostasis |
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Authors: | Bao-Zhu Yu Mahendra K. Jain Otto G. Berg |
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Affiliation: | a Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA b Department of Biophysics, IVIC, Apartado 21827, Caracas, Venezuela c Department of Molecular Evolution, Uppsala University Evolutionary Biology Center, Uppsala, Sweden |
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Abstract: | Mono- and biphasic kinetic effects of bile salts on the pancreatic IB phospholipase A2 (PLA2) catalyzed interfacial hydrolysis are characterized. This novel phenomenon is modeled as allosteric action of bile salts with PLA2 at the interface. The results and controls also show that these kinetic effects are not due to surface dilution or solubilization or disruption of the bilayer interface where in the mixed-micelles substrate replenishment becomes the rate-limiting step. The PLA2-catalyzed rate of hydrolysis of zwitterionic dimyristoylphosphatidylcholine (DMPC) vesicles depends on the concentration and structure of the bile salt. The sigmoidal rate increase with cholate saturates at 0.06 mole fraction and changes little at the higher mole fractions. Also, with the rate-lowering bile salts (B), such as taurochenodeoxycholate (TCDOC), the initial sigmoidal rate increase at lower mole fraction is followed by nearly complete reversal to the rate at the pre-activation level at higher mole fractions. The rate-lowering effect of TCDOC is not observed with the (62-66)-loop deleted ΔPLA2, or with the Naja venom PLA2 that is evolutionarily devoid of the loop. The rate increase is modeled with the assumption that the binding of PLA2 to DMPC interface is cooperatively promoted by bile salt followed by allosteric kcat?-activation of the bound enzyme by the anionic interface. The rate-lowering effect of bile salts is attributed to the formation of a specific catalytically inert E?B complex in the interface, which is noticeably different than the 1:1 EB complex in the aqueous phase. The cholate-activated rate of hydrolysis is lowered by hypolidemic ezetimibe and guggul extract which are not interfacial competitive inhibitors of PLA2. We propose that the biphasic modulation of the pancreatic PLA2 activity by bile salts regulates gastrointestinal fat metabolism and cholesterol homeostasis. |
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Keywords: | Analytical parameters are defined in Scheme A1 in Appendix A. Structures of the bile salts and conjugates are given in Figure 1 DMPC, 1,2-dimyristoyl-sn-3-glycerophospho-choline DMPM, 1,2-dimyristoyl-sn-3-glycerophospho-methanol DOC, deoxycholate DTPC, 1,2-ditetradecyl-rac-3-glycerophosphocholine i-face, the interface binding surface of PLA2 LPC, 1-myristoyl-sn-3-glycerophospho-choline MA, myristic acid MJ33, 1-hexadecyl-3-(trifluoroethyl)-rac-glycero-2-phosphomethanol PGU, 2-dodecanylamino-hexanol-2-phosphoglycol PLA2, group IB phospholipase A2 from pig pancreas POPC, 1-palmitoyl-2-oleoyl-sn-3-glycerophospho-choline products, LPC+MA (1:1) TCDOC, taurochenodeoxycholate TDOC, taurodeoxycholate TMA-DPH, trimethylammonium-diphenylhexatriene WT, wild type PLA2 |
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