Refine Active Site Model

Comparison of the actual and predicted activity of compounds in the data set is used to guide refinement of the pharmacophore and active site models. A 

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X-ray structures are determined at different levels of resolution. At low resolution only the shape of the molecule is obtained, whereas at high resolution most atomic positions can be determined to a high degree of accuracy. At medium resolution the fold of the polypeptide chain is usually correctly revealed as well as the approximate positions of the side chains, including those at the active site. The quality of the final three-dimensional model of the protein depends on the resolution of the x-ray data and on the degree of refinement. In a highly refined structure, with an R value less than 0.20 at a resolution around 2.0 A, the estimated errors in atomic positions are around 0.1 A to 0.2 A, provided the amino acid sequence is known. 

Living cells contain thousands of different kinds of catalysts, each of which is necessary to life. Many of these catalysts are proteins called enzymes, large molecules with a slotlike active site, where reaction takes place (Fig. 13.39). The substrate, the molecule on which the enzyme acts, fits into the slot as a key fits into a lock (Fig. 13.40). However, unlike an ordinary lock, a protein molecule distorts slightly as the substrate molecule approaches, and its ability to undergo the correct distortion also determines whether the key will fit. This refinement of the original lock-and-key model is known as the induced-fit mechanism of enzyme action. 

FIGURE 13.40 In the lock-and-key model of enzyme action, the correct substrate is recognized by its ability to fit into the active site like a key into a lock. In a refinement of this model, the enzyme changes its shape slightly as the key enters. 

Data analysis should focus on the development or refinement of the conceptual site model by analyzing data on source characteristics, the nature and extent of contamination, the contaminants transport pathways and fate, and the effects on human health and the environment. All field activities, sample management and tracking, and document control and inventory should be well managed and documented to ensure their quality, validity, and consistency. 

HLADH converts a wide range of substrates. For the predicition of the stereoselectivity of reduction reactions, originally Prelog s diamond lattice model was applied, which is based upon the characteristic properties of the ADH of Curvularia falcata [37]. This model describes the stereospecificity of HLADH catalyzed reductions of simple acyclic substrates such as aldehydes. Later on, for more complex acyclic and cyclic substrates, a cubic-space model of the active site was developed [38,121]. Other models are based upon symmetric properties [122-125] or upon a refined diamond lattice model [126-129]. 

Figure 11.3 Parallel-eye stereoscopic image of a model of the complete VS ribozyme (Lipfert et al., 2008). The model was constructed by connecting previously defined helical sections of a low-resolution model fitting the density map shown in Fig 11.2. Energy-minimization refinement against the standard stereochemical restraints was used to regularize and refine the structure. The scissile phosphate is shown as a sphere, and the probable active site components A756 and G638 are annotated.

Metalloenzymes pose a particular problem to both experimentalists and modelers. Crystal structures of metalloenzymes typically reveal only one state of the active site and the state obtained frequently depends on the crystallization conditions. In some cases, states probably not relevant to any aspect of the mechanism have been obtained, and in many cases it may not be possible to obtain states of interest, simply because they are too reactive. This is where molecular modeling can make a unique contribution and a recent study of urease provides a good example of what can be achieved119 1. A molecular mechanics study of urease as crystallized revealed that a water molecule was probably missing from the refined crystal structure. A conformational search of the active site geometry with the natural substrate, urea, bound led to the determination of a consensus binding model[I91]. Clearly, the urea complex cannot be crystallized because of the rate at which the urea is broken down to ammonia and, therefore, modeling approaches such as this represent a real contribution to the study of metalloenzymes. 

Lu, G., Lindqvist, Y., Schneider, G., Dwivedi, U., and Campbell, W. H., 1995, Structural studies on com nitrate reductase refined structure of the cytochrome b reductase fragment at 2.5 A, its ADP complex and an active-site mutant and modeling of the cytochrome b domain, J. Mol. Biol. 248 9319948. 

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