Enzymes, nanoconfined

In enzyme catalysis entropy is probably one of the most important factors. Enzyme reactions take place with substrates that are nanoconfined in the active sites and form a very tight enzyme-substrate complex. The catalytic groups are part of the same molecule as the substrate so there is no loss of transition or rotational entropy in the TS. 

Nanostructured materials obtained by sol-gel encapsulation of biomolecules are a novel class of biomaterials. The biological macromolecules, confined within the nanometer-size pores of the matrix, show both similarities to and differences from solution characteristics. The effects of nanoconfinement on the structural and reactivity patterns of the proteins and enzymes are discussed. The applications of these nanostructured biomaterials in the area of molecular biorecognition, detection, and biosensing are also presented. 

The paper is organized in three parts. First, the effects of nanoconfinement on the structure of the sol-gel trapped biomolecule are discussed. Second, from the results on apparent reactivity of these biomaterials, the effects of the matrix on the reactivity of trapped enzyme are elucidated. Finally, the interaction of the confined biomolecules with exogenous ligands/substrates and the applications of these materials in the area of molecular biorecognition are discussed. The reaction chemistries of biologically active molecules in die nanostructured materials have been crucial in establishing the role of the matrix upon the structure and reactivity of the confined proteins. 

Once the proteins are confmed within the porous structure of the sol-gel matrix, it becomes imperative to determine if these nanostructured materials still remain amenable to interactions with external reagents. This issue assumes central importance because the functional relevance of nanoconfined proteins and enzymes is contingent upon their reactions with suitable substrates or ligands. 

In addition to these chemical effects, physical effects due to nanoconfinement within a pore of finite dimension should adso be taken into consideration. Upon treatment of sol-gel confined enzyme with an exogenous substrate, the substrate... 

The Michaelis-Menten kinetics (75) provide a facile estimate of the altered reaction dynamics and the energetics of the nanoconfined enzyme systems. The well-known Michaelis constant Km ( k i/ki) measures the dissociation of the enzyme-substrate complex and in turn serves as an estimate of its stability. An increased value of the Michaelis constant implies that the equilibrium of the enzyme substrate complex is shifted towards the left i.e. towards the free enzyme and substrate, and suggests a relatively weaker enzyme-substrate complex. Another parameter, kcat ( k2), called the turnover number, estimates the rate of formation of the product from Ae enzyme-substrate complex. A facilitated product formation will result in increased turnover number (i.e. increased k<. The ratio kcat/ m consequently, represents the apparent rate constant for combination of a substrate with the free enzyme. The... 

Table I. Comparison of Reactivity for Nanoconfined and Free Enzymes ...

Oxalate Oxidase. For the oxalate oxidase system, the value of the Michaelis constant (Km) increased for the sol-gel nanoconfined system (Km /lO = 1.1 in solution and 4.1 in aged gel). The approximately fourfold increase indicates that the binding of the oxalate with the enzyme is weaker. The apparent association constant (kcat/ m) for the oxalate-oxalate oxidase complex was substantially altered as a result of confinement of the protein the sol-gel matrix (kcat/Km = 170 in solution and 2.3 in aged gel). This implicates a relatively destabilized enzyme-substrate complex. Additionally, the kcat parameter is also reduced in the gel (kcat /lO" = 187 in solution and 9.4 in aged gel), suggesting deactivation of the product forming step in the gel medium. In this case, whole the dissociative product forming step is reduced only by a factor of 20 (187/9.4), the associative step is reduced considerably by a factor of 75 (170/2.3). 

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