Characterization of interfacial catalysis by Aeromonas hydrophila lipase/acyltransferase in the highly processive scooting mode.
Characterization of interfacial catalysis by Aeromonas hydrophila lipase/acyltransferase in the highly processive scooting mode.
复制标题
嗜水气单胞菌脂肪酶/酰基转移酶在高度持续移动模式下的界面催化特征。
DOI：
10.1021/bi00183a003
复制
发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Gelb,MH
中科院分区：
文献类型：
--
作者：
Jain,MK;Krause,CD;Buckley,JT;Bayburt,T;Gelb,MH
Revised Manuscript Received December 10, 1993® abstract: A glycerophospholipid: cholesterol acyltransferase (GCAT) that also has lipase activity is secreted by the bacterium Aeromonas hydrophila. Hydrolysis of the in-2-ester bond of 1, 2-dimyristoyl-in-glycero-3-phosphomethanol (DMPM) vesicles by this enzyme is shown to occur in a highly processive scooting mode in which the enzyme, substrate, and the products of hydrolysis remain bound to the vesicle interface. This conclusion is based on the following observations,(a) When there is an excess of vesicles over enzyme, the hydrolysis of the jn-2-acyl group ceases after only a fraction of the total available substrate is hydrolyzed. Addition of more enzyme, but not of more substrate, leads to a new round of hydrolysis,(b) The extent of hydrolysis of vesicles per enzyme increases with the size of the vesicles, and it corresponds to the total hydrolysis of the outer monolayer of one vesicle by one enzyme,(c) The enzyme bound to vesicles composed of reaction products or of the non-hydrolyzable phospholipid 1, 2-ditetradecyl-sn-glycero-3-phosphomethanol (DTPM) is not able to undergo intervesicle exchange. Instead, intervesicle transfer of the substrate or the bound enzyme due to vesicle fusion promotes hydrolysis of all of the vesicles present in the reaction mixture,(d) Additionof DTPM vesicles to a reaction mixture containing DMPM substrate vesicles and the enzyme has no noticeable effect on the course of hydrolysis. Substrate specificity studies in the scooting mode on DMPM vesicles reveal that GCAT displays essentially no selectivity in the hydrolysis of phospholipids with different polar head groups. Treatment of GCAT withtrypsin, which removes a small peptide, results in an enzyme that displays comparable catalytic activity but increased affinity for the interface. Alkyltri-fluoromethyl ketones are shown to be tight-binding competitive inhibitors of GCAT. The scooting mode analysis, which has previously been shown toprovide a simplified approach for analyzing thesteady-state kinetics of interfacial catalysis by secreted phospholipase A2, is also useful for analyzing the interfacial kinetic behavior of lipases.The concentration of naturally-occurringphospholipids as solitary monomers in theaqueous phase is very low (Cevc & Marsh, 1987; Jain, 1988) because the hydrophobic effect on such amphiphiles promotes formation of aggregates such as vesicles, micelles, or emulsions. For such reasons, lipolytic enzymes have evolved tocarry out interfacial catalysis with great efficiency, whereas solitary substrate molecules in the aqueous phase are relatively poor substrates. As a class, lipases tend to act rather nonspecifically on ester linkages and display a preference for thesubstrate at the interface (Waite, 1987). Those lipases that have been characterized are serine esterases with no divalent cation requirement. Among lipases, the glycerophospholipid: cholesterol acyltransferase (GCAT) 1 se-creted by Aeromonas hydrophila is particularly interesting. GCAT transfers the sn-2-acyl chain of phospholipids to cholesterol (Buckley etal., 1982; Buckley, 1982, 1983). This transacylation reaction is essentially identical to thatof the well-known enzyme lecithin: cholesterol acyl transferase of mammalian plasma. In addition, GCAT hydrolyzes the sn-
