Substrate accessibility

Liidemann et al., 1997] Liidemann, S. K., Carugo, O., and Wade, R. C. Substrate access to cytochrome P450cam A comparison of a thermal motion pathway analysis with moleculM dynamics simulation data. J. Mol. Model. 3 (1997) 369-374... 

Hurst (19) discusses the similarity in action of the pyrethrins and of DDT as indicated by a dispersant action on the lipids of insect cuticle and internal tissue. He has developed an elaborate theory of contact insecticidal action but provides no experimental data. Hurst believes that the susceptibility to insecticides depends partially on the cuticular permeability, but more fundamentally on the effects on internal tissue receptors which control oxidative metabolism or oxidative enzyme systems. The access of pyrethrins to insects, for example, is facilitated by adsorption and storage in the lipophilic layers of the epicuticle. The epicuticle is to be regarded as a lipoprotein mosaic consisting of alternating patches of lipid and protein receptors which are sites of oxidase activity. Such a condition exists in both the hydrophilic type of cuticle found in larvae of Calliphora and Phormia and in the waxy cuticle of Tenebrio larvae. Hurst explains pyrethrinization as a preliminary narcosis or knockdown phase in which oxidase action is blocked by adsorption of the insecticide on the lipoprotein tissue components, followed by death when further dispersant action of the insecticide results in an irreversible increase in the phenoloxidase activity as a result of the displacement of protective lipids. This increase in phenoloxidase activity is accompanied by the accumulation of toxic quinoid metabolites in the blood and tissues—for example, O-quinones which would block substrate access to normal enzyme systems. The varying degrees of susceptibility shown by different insect species to an insecticide may be explainable not only in terms of differences in cuticle make-up but also as internal factors associated with the stability of oxidase systems. 

Adsorption on solid matrices, which improves (at optimal protein/support ratios) enzyme dispersion, reduces diffusion limitations and favors substrate access to individual enzyme molecules. Immobilized lipases with excellent activity and stability were obtained by entrapping the enzymes in hydrophobic sol-gel materials [20]. Finally, in order to minimize substrate diffusion limitations and maximize enzyme dispersion, various approaches have been attempted to solubilize the biocatalysts in organic solvents. The most widespread method is the one based on the covalent linking of the amphiphilic polymer polyethylene glycol (PEG) to enzyme molecules [21]. 

AtCCD7 (Schwartz et al. 2004). Organic solvent addition (dioxane, DMSO, methanol or acetone) improved activity under low concentrations (Mathieu et al. 2007). Short chain aliphatic alcohols activated the enzymes although the reason for this activation is unclear (probably due to influences on substrate accessibility or micellar structure). An increase in activity was observed for all aliphatic alcohols tested, although the optimal concentration lessened with increasing log P values (Schilling etal. 2007). 

The mechanisms which underlie enzyme inhibition are described more fully in Chapter 3. Suffice to say here that reversible inhibitors which block the active site are called competitive whilst those which prevent release of the product of the reaction are non-competitive. By preventing the true substrate accessing the active site, competitive inhibitors increase Km (designated by or K PParent). A non-competitive inhibitor decreases V mprime symbol ( ) here to imply physiological as it does for energy change. 

Competitive inhibitors increase Km by preventing substrate access to the active site for binding. 

Substrate Access and Product Egress Through y9-propellers... 

The energy required to activate hydrogenase is in the order of 80kJ/mol. Several possible processes have been suggested so far, e.g. a rearrangement of the spatial conformation of the active site or of the surrounding protein in order to allow substrate accessibility to the active site. 

Krainev AG, Weiner LM, Kondrashin SK, Kanaeva IP, Bach-manova GI. 1991. Substrate access channel geometry of soluble and membrane-bound cytochromes P450 as studied by interactions with type II substrate analogues. Arch Biochem Biophys 288 17-21. 

Nakayama K, Puchkaev A, Pikuleva lA. 2001. Membrane binding and substrate access merge in cytochrome P450 7A1, a key enzyme in degradation of cholesterol. J Biol... 

The enhanced catalatic activity could arise from more facile exhaust of products just as easily as from enhanced substrate accessibility. The effect of inhibitors is a largely static process that is complete once the inhibitor has become bound in the active site. The catalatic process, on the other hand, requires a constant influx of substrate peroxide and efflux of product oxygen and water. As a result, the inlet channels for inhibitors and substrate may be different. 

Fig. 5. Stereo view showing the substrate access channel in eNOS. The heme is lightly shaded and the substrate, L-Arg, is darkly shaded. The channel is deep yet solvent accessible for ready entry of substrate and exit of product. Part of the access channel is shaped by the second molecule (shaded) in the dimer. There appears to be no requirement for major structural changes upon substrate binding or release.

A further example of the use of a chiral anion in conjunction with a chiral amine was recently reported by Melchiorre and co-workers who described the asymmetric alkylation of indoles with a,P-unsaturated ketones (Scheme 65) [212]. The quinine derived amine salt of phenyl glycine (159) (10-20 mol%) provided the best platform with which to perform these reactions. Addition of a series of indole derivatives to a range of a,P-unsaturated ketones provided access to the adducts with excellent efficiency (56-99% yield 70-96% ee). The substrates adopted within these reactions is particularly noteworthy. For example, use of aryl ketones (R = Ph), significantly widens the scope of substrates accessible to iminium ion activation. Expansion of the scope of nucleophiles to thiols [213] and oximes [214] with similar high levels of selectivity suggests further discoveries will be made. 

The water-shell-model, strictly speaking, will only apply to very hydrophilic enzymes which do not contain hydrophobic parts. Many enzymes, like lipases, are surface active and interact with the internal interface of a microemulsion. In fact, lipases need a hydrophobic surface in order to give the substrate access to the active site of the enzyme. Nevertheless, Zaks and Klibanov found out that it is often not necessary to have a monolayer of water on the enzyme surface in order to perform a catalytic reaction in an organic solvent [98]. 

Figure 2.6 Schematic representation of the heme group orientation and different substrate accessibility in the heme active sites in monoxygenases and peroxidases.

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