Substrates interfacial enzyme binding

The active site of serine proteases is characterized by a catalytic triad of serine, histidine, and aspartate. The mechanism of lipase action can be broken down into (i) adsorption of the lipase to the interface, responsible for the observed interfacial activation (ii) binding of substrate to enzyme (iii) chemical reaction and (iv) release of product(s). 

Figure 10.1. Binding of an interfacial enzyme to a substrate interface. Upon binding, the enzyme adopts an interfacial conformation. The kinetics of binding is described by the rate constants of binding (kon) and dissociation (koff).

Cutinase is a hydrolytic enzyme that degrades cutin, the cuticular polymer of higher plants [4], Unlike the oflier lipolytic enzymes, such lipases and esterases, cutinase does not require interfacial activation for substrate binding and activity. Cutinases have been largely exploited for esterification and transesterification in chemical synthesis [5] and have also been applied in laundry or dishwashing detergent [6]. 

MICELLAR SUBSTRATES. Phospholipids in micelles are frequently found to be more active substrates in lipolysis than those phospholipids residing in a lipid bilayer". Dennis first described the use of Triton X-100 to manipulate the amount of phospholipid per unit surface area of a micelle in a systematic analysis of the interfacial interactions of lipases with lipid micelles. Verger and Jain et al have presented cogent accounts of the kinetics of interfacial catalysis by phospholipases. The complexity of the problem is illustrated in the diagram shown in Fig. 2 showing how the enzyme in the aqueous phase can bind to the interface (designated by the asterisk) and then become activated. Once this is achieved, E catalyzes conversion of S to release P. ... 

Although many biochemical reactions take place in the bulk aqueous phase, there are several, catalyzed by hydroxynitrile lyases, where only the enzyme molecules close to the interface are involved in the reaction, unlike those enzyme molecules that remain idly suspended in the bulk aqueous phase [6, 50, 51]. This mechanism has no relation to the interfacial activation mechanism typical of lipases and phospholipases. Promoting biocatalysis in the interface may prove fruitful, particularly if substrates are dissolved in both aqueous phases, provided that interfacial stress is minimized. This approach was put into practice recently for the enzymatic epoxidation of styrene [52]. By binding the enzyme to the interface through conjugation of chloroperoxidase with polystyrene, a platform that protected the enzyme from interfacial stress and minimized product hydrolysis was obtained. It also allowed a significant increase in productivity, as compared to the use of free enzyme, and simultaneously allowed continuous feeding, which further enhanced productivity. 

The location of the acyl chain is of primary importance in the binding process because of its size. Due to the movement of lid during interfacial activation, a hydrophobic trench is created between the lid and enzyme surface. The trench size is ideal to accommodate the acyl chain. Interactions between the non-polar residues of the trench and the non-polar acyl chain stabilize the coupling. It has been postulated that the configuration of the trench is responsible for substrate specificity. This hypothesis seems plausible since lipases usually discriminate against certain acyl chain lengths, degrees of unsaturation, and location of double bonds in the chain. Any of these factors could affect the interaction between the acyl chain and the trench. 

As discussed above in Chapter 3, ellipsometry and quartz crystal microbalance (QCM) approaches provide a useful insight into the adsorption of both the supporting interfacial assembly and the proteins themselves. Beyond monitoring the adsorption dynamics and the structural integrity of the biomolecule, the orientation of the active site is of prime importance. For example, if the active site itself binds to the self-assembled monolayer, transport of the substrate or co-enzyme may be blocked. 

Resulting from this unique potential, wide-held approaches are generally more appropriate for the study of enzymes that bind to high molecular weight substrates, such as DNA or carbohydrates. Furthermore, confocal approaches are less suitable for insoluble substrates, such as phospholipid bilayers. In this case the substrate can be considered as immobilized and the movement of the enzyme on the substrate needs to be detected. For these interfacial reactions, the catalytic step must be preceded by dihusion of the enzyme to an appropriate site on its target substrate. 

The carboxyl-terminal extension of class II enzymes forms a hemicir-cular bannister around the calcium-binding loop. It is secured proximally (Cys-126 = Cys-27) and distally (Cys-134 = Cys-50) by disulfide bridges. The 7- or 8-residue loop is rich in prolines and charged residues. This substructure is remote from the residues implicated in interfacial ad-sorpdon, substrate binding, and catalysis and has no defined catalytic or pharmacological role. 

Electroanalyhcal techniques (also in combination with other techniques, e.g., ophcal techniques such as photometry and Raman spectrometry) can be employed to inveshgate many functional aspects of proteins and enzymes in particular. It is possible to study the biocatalytic process with respect to the chemistry of the active site, the interfacial and intramolecular ET, slow enzyme achva-tors or inhibitors, the pH dependence, the transport of tlie substrate, and even more parameters. For example, slow scan voltammetry can be used to determine the relation of ET rates or of protonation and ligand binding. In contrast, fast scan voltammetry allows the determination of rates of interfacial ET. In addition, it is also possible to investigate chemical reactions that are coupled to the ET process, such as protonation. The use of direct ET for mechanistic studies of redox enzymes was recently reviewed by Leger and Bertrand [27]. Mathemahcal models help to elucidate the impact of different variables on the enhre current signal [27, 75, 76]. 

Lipases are interphase-active enzymes with hydrophobic domains. The hydro-phobic surface (loop) on lipase is thought to enable lipophilic interfacial binding with substrate molecules that actually induces the conformational changes in lipases. The open conformation will provide substrate with access to the active site, and vice versa. In certain types of lipases, the movements of a short a-hehcal hydrophobic loop in the lipase structure cause a conformational change that exposes the active sites to the substrate. This movement also increases the nonpolarity of the surface surrounding the catalytic site [30, 32, 34, 35]. Obviously, the hydrophobic surface plays an important role in the activity of lipase as an enzyme. 

When interfacial electron exchange rate(s) are sufficiently high and the response is free from mass transport hmitations, the catalytic current will be determined by the inherent activity of the enzyme. Variation of current (activity) with potential can be explained by an extension of the Michaelis-Menten description of enzyme kinetics that relates activity to oxidation state through incorporation of the Nemst equation." " The resulting expressions describe the catalytic cycle, and include rates of intramolecular electron exchange, chemical events, substrate binding and product release, together with the reduction potentials of centres in the enzyme, and the influence of... 

The nature of interfacial binding and the rate enhancements that are achieved are controversial areas. However, it is clear that both polar and non-polar interactions are involved [6] and the precise contribution of each must depend on the nature of the phospholipid interface and the interfacial binding surface of the phospholipase. A number of factors can be considered that could make a major contribution to the enhanced hydrolysis at interfaces and these relate primarily either to the substrate or to the enzyme. 

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