Enzyme potential surfaces calibration

The entropic hypothesis seems at first sight to gain strong support from experiments with model compounds of the type listed in Table 9.1. These compounds show a huge rate acceleration when the number of degrees of freedom (i.e., rotation around different bonds) is restricted. Such model compounds have been used repeatedly in attempts to estimate entropic effects in enzyme catalysis. Unfortunately, the information from the available model compounds is not directly transferable to the relevant enzymatic reaction since the observed changes in rate constant reflect interrelated factors (e.g., strain and entropy), which cannot be separated in a unique way by simple experiments. Apparently, model compounds do provide very useful means for verification and calibration of reaction-potential surfaces... 

As stated above, reliable studies of enzyme catalysis require accurate results for the difference between the activation barriers in enzyme and in solution. The early realization of this point led to a search for a method that could be calibrated using experimental and theoretical information of reactions in solution. It also becomes apparent that in studies of chemical reactions, it is more physical to calibrate surfaces that reflect bond properties (i.e., valence bond-based (VB-based) surfaces) than to calibrate surfaces that reflect atomic properties (e.g., molecular orbital-based surfaces). Furthermore, it appears to be very advantageous to force the potential surfaces to reproduce the experimental results of the broken fragments at infinite separation in solution. This can be easily accomplished with the VB picture. The resulting EVB method has been discussed extensively elsewhere,21 22 but its main features will be outlined below, because it provides the most direct microscopic connection to concepts of physical organic chemistry. 

Acetylcholineesterase and choline oxidase Carbon-fiber micro-electrode for determination of ACh and choline, mounted in a glass capillary tube. Enzyme was immobilized on the surface of the carbon fiber with albumin. Electrode was dip-coated with Nafion. Evaluation of selectivity and dynamic range, at a fixed potential of 1.2 V versus Ag-AgCl. Calibration graph for ACh and Ch was rectilinear between 0.1 and 3mM. Interference from ascorbic acid was not observed in the range 0.1 to 0.3 mM. [79]... 

The SAM consists of a mixture of octadecyl mercaptan (OM) and two short chain disulfides, which form -S-CH2-CH2-CH2-COO" and -S-CH2-CH2-NH3+ on the surface. The short chain, charged modifiers may provide defects, or pockets, in the OM layer where the enzyme may adsorb through electrostatic interactions. At oxidizing potentials, the electrode generates a catalytic current at densities up to about 10 pA/cm when exposed to fructose solution. The enzyme electrode exhibits a response time well under a minute and the calibration curve is linear at fructose concentrations up to 0.8 mM. The biosensor prototype exhibits low susceptibility to positive interference by ascorbic acid indicating that this construct could be useful for fructose analysis of citrus fruit juice. 

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