Enzyme catalysis external effects

The catalytic behavior of enzymes in immobilized form may dramatically differ from that of soluble homogeneous enzymes. In particular, mass transport effects (the transport of a substrate to the catalyst and diffusion of reaction products away from the catalyst matrix) may result in the reduction of the overall activity. Mass transport effects are usually divided into two categories - external and internal. External effects stem from the fact that substrates must be transported from the bulk solution to the surface of an immobilized enzyme. Internal diffusional limitations occur when a substrate penetrates inside the immobilized enzyme particle, such as porous carriers, polymeric microspheres, membranes, etc. The classical treatment of mass transfer in heterogeneous catalysis has been successfully applied to immobilized enzymes I27l There are several simple experimental criteria or tests that allow one to determine whether a reaction is limited by external diffusion. For example, if a reaction is completely limited by external diffusion, the rate of the process should not depend on pH or enzyme concentration. At the same time the rate of reaction will depend on the stirring in the batch reactor or on the flow rate of a substrate in the column reactor. 

The term commitment to catalysis " is unique to KIE studies on enzymes and not widely used in the chemical literature. External commitment to catalysis refers to the partitioning of the hrst intermediate of the reaction, the non-covalent enzyme-substrate (E-S) intermediate. Some enzymes are such efficient catalysts that nearly every molecule of substrate that binds proceeds forward to products (i.e. fes > 2)- Under these conditions, the hrst irreversible step of the reaction is substrate binding. If competitive isotope effects are measured, they will only reflect binding isotope effects, and have no contribution from the chemical steps of the reaction. In less extreme cases (Eig. 2d), both substrate binding ( 1) and the chemical step ( 3) are partially irreversible. Internal commitment to catalysis simply refers to the partitioning of any intermediate enzyme-bound species. 

The role played by the factors that contribute to enzyme efficiency has been investigated using simple molecular models. A convenient method to study the role of proximity is to use intra-molecular models. In this context, it is useful to define the effective molarity (EM) as the (first-order) rate constant for the intra-molecular reaction divided by the (second-order) rate constant for the equivalent inter-molecular process. The EM is nominally the concentration of the equivalent external catalyst needed to make the inter-molecular rate match that of the intra-molecular reaction [10]. An example of intra-molecular acid catalysis was presented in Figure 13.8a, in the context of the hydrolysis of aspirin. 

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