Protein crystallography identification

A critical input in unraveling the catalytic mechanism of epoxide hydrolases has come from the identification of essential residues by a variety of techniques such as analysis of amino acid sequence relationships with other hydrolases, functional studies of site-directed mutated enzymes, and X-ray protein crystallography (e.g., [48][53][68 - 74]). As schematized in Fig. 10.6, the reaction mechanism of microsomal EH and cytosolic EH involves a catalytic triad consisting of a nucleophile, a general base, and a charge relay acid, in close analogy to many other hydrolases (see Chapt. 3). 

For protein crystallography, the repository of most protein crystal structures is the PDB hosted at http // www.rcsb.org/pdb/ (Berman et al., 2000). This database contains the 3-D coordinates (and sometimes the structure factor files) for almost all protein crystal structures. Most journals currently require deposition of the coordinates when pubhshing stmcture papers. Each structure is given a unique identification code that will be listed in the paper (see Figure 22-1 for examples of PDB codes). Structures can be accessed using this code, or using various other search criteria. The PDB also contains structural information for NMR structures. 

Advances in protein crystallography have been responsible for major insights into the structures of Mo and related W enzymes. Early results confirmed the essential structure (in two forms) and dithiolenic coordination mode of MPT proposed by Rajagopalan and coworkers. Crystal structures of D. gigas AOR revealed a five-coordinate, square-pyramidal active site but the identification of an apical sulfido ligand remained contentious. Nevertheless, MoHMs of the t)q)e [(DT)MoOS(OH)] (DT = dithiolene) were now squarely on the drawing board. Recently, Moura and coworkers provided evidence that D. gigas AOR is a functional dioxo-Mo(vi) hydroxylase. 

The use of crystallography for identification of key residues of proteins is a well-established technique. Crystallography merely provides a model for how these amino acids are involved in function, and biochemical tests are still required to confirm the actual roles. 

Before they were characterized by x-ray crystallography, the classification of the structures of copper proteins was initially based on the spectroscopic features of their active site in the oxidized state. The tremendous development of crystallographic and spectroscopic techniques in recent years has enabled the identification of as many as seven different types of active sites in these proteins type 1, type 2, type 3, type 4, CuA, CuB and Cuz. The characteristics of these metal sites are briefly described below. 

Group-specific chemical modification remains a useful method for studies of structure-function relationships in protein molecules, although unambiguous identification of essential amino acid residues and elucidation of their function are nowadays accomplished mainly by X-ray crystallography and site-directed mutagenesis. Chemical modifications... 

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