Surface Concentration Of Substrate Research Articles

Theoretical Analysis of Immobilized Oxidase Enzyme Electrode in the Presence of Two Oxidants

In this paper, mathematical model of Martens and Hall (Analytical chemistry 66, 2763-2770 (1994)) for an immobilized oxidase enzyme electrode is discussed. The model involves the system of non-linear reaction diffusion equations under the steady state conditions. A simple and closed-form of approximate analytical expressions for the concentrations of the immobilization of three enzyme substrates has been derived by solving the system of non-linear reaction diffusion equations using new approach of homotopy perturbation method. Approximate polynomial expression of concentration of substrate, oxygen and oxidized mediator and current was obtained in terms of the Thiele moduli and the small values of parameters Bs, Bo and Bm (normalized surface concentration of substrate, oxygen and oxidized mediator). Furthermore, in this work the numerical simulation of the problem is also reported using Matlab program. An agreement between analytical expressions and numerical results is noted.

Analytical Expressions for Steady-State Concentrations of Substrate and Oxidized and Reduced Mediator in an Amperometric Biosensor

A mathematical model of modified enzyme-membrane electrode for steady-state condition is discussed. This model contains a nonlinear term related to enzyme kinetics reaction mechanism. The thickness dependence of an amperometric biosensor is presented both analytically and numerically where the biological layer is immobilized between a solid substrate and permeable electrode. The analytical expressions pertaining to the concentration of species and normalized current are obtained using the Adomian decomposition method (ADM). Simple and approximate polynomial expressions of concentrations of an oxidized mediator, substrate, and reduced mediator are derived for all possible values of parametersϕO2(Thiele modulus),BO(normalized surface concentration of oxidized mediator), andBS(normalized surface concentration of substrate). A comparison of the analytical approximation and numerical simulation is also presented. A good agreement between theoretical predictions and numerical results is observed.

REGULATION OF DIACYLGLYCEROL KINASE THETA

Diacylglycerol (DAG) and phosphatidic acid are important signaling molecules involved in numerous signaling pathways. The diacylglycerol kinases (DGKs) are particularly poised to regulate the cellular levels of these two lipids. Although interest in these enzymes is increasing, information regarding their regulation at a molecular level is scarce, particularly information pertaining to the enzymology of these enzymes. We recently showed that DGK‐θ behaves a “quasi‐scooter” given its sensitivity to both the bulk (total) and surface concentration of substrate. Additionally, stimulation of the cultured fibroblasts expressing this enzyme leads to an increase in the apparent Km (Kmapp) which was decreased in response to an increase in the bulk substrate (DOG, dioleylglycerol) concentration. Further progress on understanding the mammalian DGKs at a molecular level have been hindered in large part by the paucity of purified DGKs with significant catalytic activity. To this end, we have purified DGK‐θ to apparent homogeneity. Interestingly, the activity of purified DGK‐θ is extremely weak. Interestingly, addition of certain proteins results in a dramatic increase in its activity in vitro. Studies designed to characterize these activating proteins and their effects on the enzymology of DGK‐θ are in progress. One of the major challenges of this research will be to identify the physiologically relevant activating protein(s) and work is in progress to accomplish this goal.

Signaling at the membrane interface by the DGK/SK enzyme family

The sphingosine (SK) and diacylglycerol (DGK) kinases have become the subject of considerable focus recently due to their involvement as signaling enzymes in a variety of important biological processes. These lipid signaling kinases are closely related by sequence as well as functional properties. These enzymes are soluble, yet their substrates are hydrophobic. Therefore, they must act at the membrane interface. Second, for both of these enzyme families, their substrates (diacylglycerol for DGKs, sphingosine for SKs) as well as their products (phosphatidic acid for DGK, sphingosine-1-phosphate for SK) have signaling function. To understand how the signaling processes emanating from these kinases are regulated it is critical to understand the fundamental mechanisms that control their enzymatic activity. This is particularly true for the rational design of small molecules that would be useful as therapeutic compounds. Here we summarize enzymological properties of the diacylglycerol and SKs. Further, because the three-dimensional structure of the eukaryotic members of this family has yet to be determined, we discuss what can be gleaned from the recently reported structures of related prokaryotic members of this enzyme family.

Analysis of a Novel Diacylglycerol Kinase from Dictyostelium discoideum: DGKA

Diacylglycerol kinases (DGKs) catalyze the ATP-dependent phosphorylation of diacylglycerols to generate phosphatidic acid and have been investigated in prokaryotic and eukaryotic organisms. Recently, a protein that is significantly similar to human DGK-theta, DGKA, was identified in Dictyostelium discoideum. It has been shown to possess DGK activity when assayed using a medium-chain diacylglycerol, 1,2-dioctanoyl-sn-glycerol (DiC8). A complete understanding of DGK catalytic and regulatory mechanisms, as well as physiological roles, requires an understanding of its biochemical and kinetic properties. This report presents an analysis of these properties for DGKA. The enzyme catalyzes the phosphorylation of DiC8, and another medium-chain DAG, DiC6 (1,2-dihexanoyl-sn-glycerol), in a Michaelis-Menten manner. Interestingly, the kinetics of DGKA using physiologically relevant long-chain DAGs was dependent on substrate surface concentration and the detergent that was used. DGKA displayed Michaelis-Menten kinetics with respect to bulk substrate concentration (1,2-dioleoyl-sn-glycerol) in octyl glucoside mixed micelles when the surface substrate concentration was at or below 3.5 mol %. At higher surface concentrations, however, there was a sigmoidal relationship between the initial velocity and bulk substrate concentration. In contrast, DGKA displayed sigmoidal kinetics with respect to bulk substrate concentrations at all surface concentrations in Triton X-100 mixed micelles. Finally, we show the catalytic activity of DGKA was significantly enhanced by phosphatidylserine (PS) and phosphatidic acid (PA).

Action of Phosphatidylinositol-specific Phospholipase Cγ1 on Soluble and Micellar Substrates: SEPARATING EFFECTS ON CATALYSIS FROM MODULATION OF THE SURFACE

The kinetics of PI-PLCgamma1 toward a water-soluble substrate (inositol 1,2-cyclic phosphate, cIP) and phosphatidylinositol (PI) in detergent mixed micelles were monitored by 31P NMR spectroscopy. That cIP is also a substrate (Km = approximately 15 mM) implies a two-step mechanism (intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)). PI is cleaved by PI-PLCgamma1 to form cIP and I-1-P with the enzyme specific activity and ratio of products (cIP/I-1-P) regulated by assay temperature, pH, Ca2+, and other amphiphilic additives. Cleavage of both cIP and PI by the enzyme is optimal at pH 5. The effect of Ca2+ on PI-PLCgamma1 activity is unique compared with other isozymes enzymes: Ca2+ is necessary for the activity and low Ca2+ activates the enzyme; however, high Ca2+ inhibits PI-PLCgamma1 hydrolysis of phosphoinositides (but not cIP) with the extent of inhibition dependent on pH, substrate identity (cIP or PI), substrate presentation (e.g. detergent matrix), and substrate surface concentration. This inhibition of PI-PLCgamma1 by high Ca2+ is proposed to derive from the divalent metal ion-inducing clustering of the PI and reducing its accessibility to the enzyme. Amphiphilic additives such as phosphatidic acid, fatty acid, and sodium dodecylsulfate enhance PI cleavage in micelles at pH 7.5 but not at pH 5.0; they have no effect on cIP hydrolysis at either pH value. These different kinetic patterns are used to propose a model for regulation of the enzyme. A key hypothesis is that there is a pH-dependent conformational change in the enzyme that controls accessibility of the active site to both water-soluble cIP and interfacially organized PI. The low activity enzyme at pH 7.5 can be activated by PA (or phosphorylation by tyrosine kinase). However, this activation requires lipophilic substrate (PI) present because cIP hydrolysis is not enhanced in the presence of PA.

Kinetics of Bacillus cereus phosphatidylinositol-specific phospholipase C with thiophosphate and fluorescent analogs of phosphatidylinositol.

Thiophosphate analogs (C-S-P bond) of phosphatidylinositol (Cn-thio-PI: racemic hexadecyl-, dodecyl-, and octylthiophosphoryl-1-myo-inositol) and a fluorescent analog (pyrene-PI: rac-4-(1-pyreno)-butylphosphoryl-1-myo-inositol) were all substrates for phosphatidylinositol-specific phospholipase C from Bacillus cereus. Hydrolysis of thio-PI was followed by coupling the production of alkylthiol to a disulfide interchange reaction with dithiobispyridine. Hydrolysis of pyrene-PI was followed using a HPLC-based assay with fluorescence detection. The activity of PI-PLC with thio-PI analogs showed an interfacial effect. C16-Thio-PI, which had a critical micelle concentration (CMC) of 7 microM, gave a hyperbolic activity versus concentration curve between 0 and 2 mM, while C8-thio-PI, which had a CMC above 10 mM, showed very low activity which increased greatly upon introduction of an interface in mixed micelles with hexadecylphosphocholine (HDPC). Pyrene-PI, which aggregates above 0.3 mM, gave a sigmoidal activity curve with much higher activity above the CMC. All three thio-PI homologs as mixed micelles with HDPC gave hyperbolic activity curves with PI-PLC that were a function of bulk concentration of substrate at constant surface concentration and surface concentration of substrate at constant bulk concentration. The maximal activity of PI-PLC with pure C16-thio-PI micelles was 6.25 mumol min-1 mg-1, while that with pyrene-PI was estimated to be 68 mumol min-1 mg-1. With pure C16-thio-PI micelles, 0.022 mM substrate gave half Vmax, similar to that in mixed micelles with HDPC.

Cinétique de l'hydrolyse par une lipase d'un glycéride étalé en couche monomoléculaire: I. — Influence de la concentration en enzyme et de la concentration superficielle du substrat

Summary The variation with pH of the rate of surface hydrolysis of dicaprin shows the same maxima and minima as shown by lipase in ordinary conditions. For a given surface concentration of substrate, the phenomenon of saturation (i.e. constant maximum of reaction rate) is observed above a certain concentration of enzyme. Varying the substrate surface concentration (or molecular area), sudden abrupt changes are observed in the rate of reaction for some particular values of the molecular area. These changes of slope are apparently connected to changes in the molecular conformation of the substrate.