Site-directed mutagenesis enzyme structure

Structural details are also available for the class II (/ia)8-barrel enzyme FruA from E. coli at excellent resolution (1.6A) [51, 52]. The homodimeric protein requires movement of the divalent zinc cofactor from a buried position to the catalytically effective surface position. Recent attempts to explore the origin of substrate discrimination of the structurally related E. coli aldolase vith specificity for tagatose 1,6-bisphosphate by site-directed mutagenesis and structure determinations highlight the complexity of enzyme catalysis in this class of enzymes and the subtleties in substrate control [53-55]. 

The lack of structural similarity between a feedback inhibitor and the substrate for the enzyme whose activity it regulates suggests that these effectors are not isosteric with a substrate but allosteric ( occupy another space ). Jacques Monod therefore proposed the existence of allosteric sites that are physically distinct from the catalytic site. Allosteric enzymes thus are those whose activity at the active site may be modulated by the presence of effectors at an allosteric site. This hypothesis has been confirmed by many lines of evidence, including x-ray crystallography and site-directed mutagenesis, demonstrating the existence of spatially distinct active and allosteric sites on a variety of enzymes. 

With these possible applications in mind, we should review the significant characteristics of hydrogenase as a catalyst. Compared with most chemical catalysts, hydrogenases are large molecules. The protein has been selected by evolution, from an almost infinite number of possible structures. The whole protein is part of the machinery. Therefore even minor tampering with the protein, for example by site-directed mutagenesis, is likely to lead to unexpected changes in the properties of the enzyme. 

Unlike the AAOs, whose very name implies a breadth of substrate specificity, the aaDHs tend to be rather more specific and are each named after their optimal amino acid substrate. In particular, the archetype in this enzyme family, GDH, shows poor activity with most substrates other than glutamate. However, the availability of X-ray crystallographic structures for several aaDHs ° has opened the door to site-directed mutagenesis and altered substrate specificities. " ... 

The proximal calcium binding site is coupled to the heme group by virtue of the fact that one of its ligands, Thrl71, is adjacent to the proximal histidine residue, Hisl70 (Fig. 4). The results of site-directed mutagenesis studies at this position are awaited with interest. An illustration of the importance of both calcium sites to the structure and function of HRP C is afforded by the need to incorporate calcium as a component of in vitro folding mixtures to obtain active recombinant enzyme from solubilized inclusion bodies (64). 

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