Enzyme site, computer modeling

Hen egg-white lysozyme catalyzes the hydrolysis of various oligosaccharides, especially those of bacterial cell walls. The elucidation of the X-ray structure of this enzyme by David Phillips and co-workers (Ref. 1) provided the first glimpse of the structure of an enzyme-active site. The determination of the structure of this enzyme with trisaccharide competitive inhibitors and biochemical studies led to a detailed model for lysozyme and its hexa N-acetyl glucoseamine (hexa-NAG) substrate (Fig. 6.1). These studies identified the C-O bond between the D and E residues of the substrate as the bond which is being specifically cleaved by the enzyme and located the residues Glu 37 and Asp 52 as the major catalytic residues. The initial structural studies led to various proposals of how catalysis might take place. Here we consider these proposals and show how to examine their validity by computer modeling approaches. 

A topic of actuality is the study of receptor proteins and enzymes for which data bases with crystallographic information are now made available. Computer modelling of the active sites of receptors and enzymes are important tools in rational drug design. Principal components and cluster analysis can be applied to the primary... 

In the absence of definitive information about the structure of the active site theoretical modeling of enzyme catalyzed reactions is difficult but not impossible. These difficulties are caused by the extremely large size of the enzyme-substrate-solvent system which typically comprises thousands or tens of thousands of atoms so that direct theoretical treatment at the microscopic quantum mechanical level is not yet practical. The computational demand is simply too enormous. As a compromise, a scheme generally referred to as QM/MM (quantum mechanics/molecular mechanics) has been devised. In QM/MM calculations, the bulk of the enzyme-solvent system (i.e. most of the atoms) is treated at a low cost, usually at the molecular mechanics (MM) level, while the more nearly correct and much more expensive quantum level (QM) computation is applied only to the reaction center (active site). 

The task now was to locate the natural substrate and the flexible inhibitors in a three dimensional computer model of the enzyme site to examine if interactions with these features could go some way to providing plausible structure/activity relationships. 

Fig. 1. Construction of a computational model for TauD. (A) the solvated TauD enzyme (PDB code 1GY9, solvating water molecules in red) (B) the desolvated enzyme (C) the active site with the substrate and a-ketoglutarate bound to the iron centre, and the most important amino acids in the first and second coordination sphere (D) a minimal model for TauD including only the first coordination sphere and the substrate.

Mutations can also be incorporated into the ALS gene in order to alter the specific interactions between the enzyme and its inhibitors. Site directed mutagenesis has been used to incorporate mutations into cloned plant ALS genes based both on previously characterized mutants and on computer modeling information. These altered genes have been transformed into plants, and the transgenic plants have been tested for their sensitivity to a variety of... 

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