Surface dilution kinetics using substrate analog enantiomers as diluents: Enzymatic lipolysis by bee venom phospholipase A 2 (original) (raw)
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Lipases at interfaces: A review
Advances in Colloid and Interface Science, 2009
Lipases are acyl hydrolases that play a key role in fat digestion by cleaving long-chain triglycerides into polar lipids. Due to an opposite polarity between the enzyme (hydrophilic) and their substrates (lipophilic), lipase reaction occurs at the interface between the aqueous and the oil phases. Hence, interfaces are the key spots for lipase biocatalysis and an appropriate site for modulating lipolysis. Surprisingly enough, knowledge about the effects of the interfacial composition on lipase catalysis is still limited and only described by the term "interfacial quality". Recent systematic studies based on a biophysical approach allowed for the first time to show the effects of the interfacial microenvironment on lipase catalysis. These studies demonstrate that lipase activity as a function of interfacial composition is more attributed to substrate inaccessibility rather than to enzyme denaturation or inactivation, as it is often hypothesized. A detailed analysis of the interfacial properties of all compounds involved in triglyceride digestion revealed that lipolysis is a self-regulated reaction. This feedback mechanism can be explored as a new avenue to control lipase catalysis. To substantiate this hypothesis, oil hydrolysis in a model gastro-intestinal system was performed, which can be seen as an interfacial engineering approach to enzyme reactivity control. The presented characterization of the interfacial composition and its consequences provide a new approach for the understanding of lipase reactions at interfaces with direct impact on biotechnological and health care applications.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015
Surface pressure Isochoric method Bothrops diporus sPLA 2 Bothrops asper myotoxin We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Π opt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Π opt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA 2 s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA 2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA 2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Π opt , validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN•m −1 and 10 mN•m −1 , termed here as LR 20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA 2 s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A 2 .
Biochimica Et Biophysica Acta Lipids and Lipid Metabolism, 1989
A rapid, continuous spectrophotometric assay for measuring the amount and activity of several |ipolytic enzymes is described. It is based on the metachromatic properties of the cationic dye safranine, and makes use of the fact that an adequate combination of a iipolytic enzyme with one of its substrates leads to a change in the net negative charge at the lipid/water interface, which is monitored by the absorbanee change of safranine. Utilizhig this method, most lilmlytic enzymes can be detected in very low amounts (mUliunR or less) in about I rain without employing radiolabel|ed lipids or synthetic lipid analogues. Over a wide range of enzyme concentrations, there is a good lineariff between the initial hydrolysis rate (determination by the safranine method) and the amount of enzyme. The versatility of the assay is illustrated by examples showing how phospholipase A 2, triacyiglycerol hydrolase, phospholipase D or phospholipase C (either general or pbosphatidylinositol-specific) activities can be detected, either separately or sequentially. Due to its high sensitivity, simplicity and rapidity, this assay should find its main application in monitoring column effluents during the purification steps of iipolytic enzymes.
Effect of the lipid hydrolysis products on the phospholipase A2 action towards lipid monolayer
Chemistry and Physics of Lipids, 1994
The effect of laurie acid (LA) and lysolauroyllecithin (LLL) on the hydrolysis of lipid in monolayer by phospholipase A2 from Bee venom was studied. It was found that LLL inhibits phospholipase action under both high (39 mN/m) and low (25 raN/m) surface pressure. On the other hand, LA inhibits phospholipase action under the low surface pressure (15 mN/m or 25 raN/m), but increases enzyme activity under high surface pressure (39 raN/m). This activating effect can be suppressed by high ionic strength of the aqueous subphase. It is suggested that an increase of the negative surface charge of the lipid monolayer, followed by an increase of the local concentrations of the positively charged enzyme and calcium near the monolayer is a coupling factor between fatty acid accumulation and phospholipase activation. Such an autocatalytie process can only occur when the substrate is organised into monolayer, bilayer or micelles, therefore it can be considered as a reason for the substrate activation and induction time before lipid hydrolysis.
This review focuses on the mechanism of action of phospholipase A2 from cobra venom (Naja naja naja) toward the lipid/water interface. Particular points of interest include dramatic changes in the enzyme activity if the physical state of its substrate is altered and the activation of the enzyme by phosphorylcholine containing lipids. The experimental findings include the following: Micellar substrates are hydrolyzed faster by the enzyme than various bilayer forms of substrate aggregation. The activity of the enzyme toward short chain phospholipids increases suddenly above their critical micelle concentrations. An abrupt change in susceptibility to the enzyme is observed at the thermotropic phase transition of phospholipid vesicles. The enzyme shows the kinetic phenomena of surface dilution and activation by certain lipids, which suggest a two-step mechanism of action. A model is discussed which accommodates the present data both for the action of this enzyme at various lipid/water interfaces as well as its interaction with synthetic monomeric ligands and substrates.