Reference: Chae M and Carman GM (2013) Characterization of the yeast actin patch protein App1p phosphatidate phosphatase. J Biol Chem 288(9):6427-37

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Abstract


Yeast App1p is a phosphatidate phosphatase (PAP) that associates with endocytic proteins at cortical actin patches. App1p, which catalyzes the conversion of phosphatidate (PA) to diacylglycerol, is unique among Mg-dependent PAP enzymes in that its reaction is not involved with de novo lipid synthesis. Instead, App1p PAP is thought to play a role in endocytosis because its substrate and product facilitate membrane fission/fusion events and regulate enzymes that govern vesicular movement. App1p PAP was purified from yeast and characterized with respect to its enzymological, kinetic, and regulatory properties. Maximum PAP activity was dependent on Triton X-100 (20 mm), PA (2 mm), Mg (0.5 mm), and 2-mercaptoethanol (10 mm) at pH 7.5 and 30 degrees C. Analysis of surface dilution kinetics with Triton X-100/PA-mixed micelles yielded constants for surface binding (K = 11 mm), interfacial PA binding (K = 4.2 mol %), and catalytic efficiency (V = 557 mumol/min/mg). The activation energy, turnover number, and equilibrium constant were 16.5 kcal/mol, 406 s, and 16.2, respectively. PAP activity was stimulated by anionic lipids (cardiolipin, phosphatidylglycerol, phosphatidylserine, and CDP-diacylglycerol) and inhibited by zwitterionic (phosphatidylcholine and phosphatidylethanolamine) and cationic (sphinganine) lipids, nucleotides (ATP and CTP), N-ethylmaleimide, propranolol, phenylglyoxal, and divalent cations (Ca, Mn, and Zn). App1p also utilized diacylglycerol pyrophosphate and lyso-PA as substrates with specificity constants 4- and 7-fold lower, respectively, when compared with PA.

Reference Type
Journal Article | Research Support, N.I.H., Extramural
Authors
Chae M, Carman GM
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