Site-directed mutagenesis structural analysis

Szklarz GD, Halpert JR. Use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships of mammalian cytochromes P450. Life Sci 1997 61 2507-20. 

Conjunction with Site-Directed Mutagenesis For Analysis of Structure-Function Relationships of Mammalian Cytochromes P450. 

Prior to the advent of site-directed mutagenesis as a viable technique for the production of specifically modified proteins, the last major event to exert a major influence on the study of protein structure and function was the development of X-ray diffraction analysis for the detailed structural analysis of macromolecules. In the intervening thirty years, the availability of protein structures obtained in this manner combined with a wide range of physical and chemical studies of these proteins allowed development of substantial insight into the relationship between the structure of a protein and its functional attributes. There was some reason to expect, therefore, that functional characterization of specifically mutated proteins based on understanding developed with more classical techniques should permit efficient confirmation of existing hypotheses, particularly for proteins for which the available literature is as extensive as that for cytochrome c. 

Site-directed mutagenesis and the crystal structure analysis of a proteasome-inhibitor complex identified the amino-terminal threonine (Thrl) of Thermoplasma P subunits as both, the catalytic nucleophile and the primary proton acceptor (Seemiiller et al. 1995 Lowe et al. 1995). 

Domanski, T. L. and Halpert, J. R. (2001) Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis. Curr. Drug. M. 2, 117-137. 

The results of site-directed mutagenesis analysis of zinc ligands of higher plant p-carbonic anhydrase and of P. purpureum carbonic anhydrase have confirmed that zinc is essential for catalysis. X-ray fine structure data indicated that a water molecule is hydrogen bonded to the zinc-ligated Asp-151 and Asp-405. The water molecule is not directly coordinated to the zinc atom. A possible catalytic mechanism of C02 hydration cycle (211) has been proposed as given in Scheme 10. 

Earlier work in this field [28] indicated that acetylcholinesterase enzymes would be suitable biomolecules for the purpose of pesticide detection, however, it was found that the sensitivity of the method varied with the type and source of cholinesterase used. Therefore the initial thrust of this work was the development of a range of enzymes via selective mutations of the Drosophila melanogaster acetylcholinesterase Dm. AChE. For example mutations of the (Dm. AChE) were made by site-directed mutagenesis expressed within baculovirus [29]. The acetylcholinesterases were then purified by affinity chromatography [30]. Different strategies were used to obtain these mutants, namely (i) substitution of amino acids at positions found mutated in AChE from insects resistant to insecticide, (ii) mutations of amino acids at positions suggested by 3-D structural analysis of the active site,... 

All nitrilases belonging to aromatic nitrilase and arylacetonitrilase are susceptible to SH-reagents, except the R. rhodochrous K22 aliphatic nitrilase [81]. Analysis by site-directed mutagenesis for our three nitrilases from R. rhodochrous J1 [83], R. rhodochrous K22 [84] and Alcaligenes faecalis JM3 [85] revealed that a unique cysteine that is conserved at the corresponding position in each nitrilase is essential for the catalytic activity (Fig. 7). All nitrilases whose structural genes are cloned have a similar amino acid sequence nitrilase forms a superfamily. 

Domanski TL, Schultz KM, Roussel F, et al. Structure-function analysis of human cytochrome P-4502B6 using a novel substrate, site-directed mutagenesis, and molecular modeling. J Pharmacol Exp Ther 1999 290 1141-1147. 

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