Enzyme chemistry functionality assessment

Clearly, the chemistry carried out on model systems for both B12-coenzyme dependent and flavin-dependent enzymes have suggested a number of mechanisms by which the natural systems might function. As with all biomimetic chemistry, the information given by the in vitro studies must be compared with and extended to the in vivo system to assess whether or not the model chemistry bears any relationships to the natural systems. 

So far, we have summarized strategies to exploit the chemical versatility of polymer brushes to either immobilize biomolecules by covalent attachment or for significantly decreasing protein adsorption. However, the extended interface created by the brush in a good solvent also provides a swellable, soft layer that can promote the nonspecific immobilization of enzymes and provide an environment that supports their activity. We have tested the functionality of enzymes physisorbed from solution [11]. Because this type of binding is weak, the conformation and activity of the proteins is expected to remain largely intact. To assess the influence of polymer brush chemistry, wettability, and swellability on the physisorption of proteins, model enzymes were chosen. Alkaline phosphatase (ALP) and horseradish peroxidase (HRP) were selected because they both catalyze the transformation of a colorless substrate to a colored product, and the enzymatic activity can therefore be easily monitored with colorimetry. The substrate of choice for ALP is /lara-nitrophenyl phosphate (pNPP), which is hydrolyzed to yield yellow /lara-nitrophenol (pNP) (Figure 4.14). 

As medicinal chemistry teams began to design and synthesize new NS3 protease inhibitors, a preliminary assessment of inhibitor potency was accomplished using a functional biochemical enzyme assay. The eventual development of a cell-based HCV replicon assay enabled the assessment of new inhibitors in a more physiologically cellular environment, thus providing a major advance in discovery efforts for this target. 

Besides the classical techniques for structural determination of proteins, namely X-ray diffraction or nuclear magnetic resonance, molecular modelling has become a complementary approach, providing refined structural details [4—7]. This view on the atomic scale paves the way to a comprehensive smdy of the correlations between protein structure and function, but a realistic description relies strongly on the performance of the theoretical tools. Nowadays, a full size protein is treated by force fields models [7-10], and smaller motifs, such as an active site of an enzyme, by multiscale approaches involving both quantum chemistry methods for local description, and molecular mechanics for its environment [11]. However, none of these methods are ab initio force fields require a parameterisation based on experimental data of model systems DPT quantum methods need to be assessed by comparison against high level ab initio calculations on small systems. 

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