Substrate efflux propensity plays a key role in the specificity of secretory A-type phospholipases.
To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9-10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA(2)s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA(2)s are key players.
Haimi
Perttu
P
Department of Medical Biochemistry and Developmental Biology, Institute of Biomedicine, University of Helsinki, PL 63, 00014 Helsinki, Finland.
Hermansson
Martin
M
Batchu
Krishna Chaithanya
KC
Virtanen
Jorma A
JA
Somerharju
Pentti
P
D000818:Animals; D005960:Glucosides / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006868:Hydrolysis; D008051:Lipid Bilayers; D013058:Mass Spectrometry; D008823:Micelles; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D034421:Sus scrofa; D053835:Unilamellar Liposomes / Q000378:metabolism
D005960:Glucosides; D008051:Lipid Bilayers; D008823:Micelles; D010713:Phosphatidylcholines; D053835:Unilamellar Liposomes; D010741:Phospholipases A