Directional mutation

We know much more about factors that influence the stability of the native state, mainly from experiments using directed mutations in proteins of known three-dimensional structure. Such experiments have yielded... 

Matthews, B.W., Nicholson, H., Becktel, WJ. Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc. Natl. 

Common to all three aims is that silico-derived predictions can rationalize experimental efforts either by well-directed very specific molecular biological experiments like site directed mutations or e.g., by reducing the number of compounds to screen experimentally for drug design. 

Fig. 3 Activity of 1, 2, and 3 wild type HCV NS5B and site-directed mutations. Binding and...

Thomas AW, J Lewington, S Hope, AW Topping, AJ Weightman, JH Slater (1992) Environmentally directed mutations in the dehalogenase system of Pseudomonas putida strain PP3. Arch Microbiol 158 176-182. 

Thus it may prove possible to improve O2 tolerance further by directed mutation of residues within the protein. 

Kitayama, S., Shimada, S., Xu, H., Markham, L., Donovan, D.M., and Uhl, G.R., Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding, Proc. Natl. Acad. Sci. U.S.A., 89, 7782, 1992. 

Ho, B.Y., Karschin, A., Branchek, T., Davidson, N. and Lester, H.A. (1992) The role of conserved aspartate and serine residues in ligand binding and in function of the 5-HT1A receptor a site-directed mutation study. FEBS Letters, 312, 259-262. 

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). 

We have used a series of biocatalysts produced by site-directed mutations at the active site of L-phenylalanine dehydrogenase (PheDH) of Bacillus sphaericus, which expand the substrate specificity range beyond that of the wild-type enzyme, to catalyse oxidoreduc-tions involving various non-natural L-amino acids. These may be produced by enantiose-lective enzyme-catalysed reductive amination of the corresponding 2-oxoacid. Since the reaction is reversible, these biocatalysts may also be used to effect a kinetic resolution of a D,L racemic mixture. ... 

Several site-directed mutations were carried out with a new member of the blue oxidases , the Fet3p protein from Saccharomyces cerevisiae which is involved in iron trafficking. The protein oxidizes Fe2+ to Fe3+ as first step in the iron uptake chain. The mutations were targeted to block this ferroxidase activity and thus to identify the residues involved in iron binding. The respective mutants showed no perturbed type 1 sites.91... 

The dilferent EPR properties (g) values, relaxation behaviour) of Fj and Fj-together with site-directed mutations allowed the assignment of the 2 clusters to specific sites in the PS I protein.325-326 This assignment was found to be the same in PS I from different species.188... 

Since completion of this chapter, two additional papers on site-directed mutations of anthopleurin-B have been published. In the first (Khera PK, Blumenthal... 

Miles, R.D. Iyer, P.P. Ferry, J.G. Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila. J. Biol. Chem., 276, 45059-45064 (2001)... 

Strong, S.J. Ellington, W.R. Expression of horseshoe crab arginine kinase in Escherichia coli and site-directed mutations of the reactive cysteine peptide. Gomp. Biochem. Physiol. B, 113, 809-816 (1996)... 

Direct mutation in the Rb gene may also inactivate the protein product. Alternatively, the Rb promoter can be methylated and so inactivated. 

Impressed by the specificity of enzymatic action, biochemists early adopted a "lock-and-key" theory which stated that for a reaction to occur the substrate must fit into an active site precisely. Modem experiments have amply verified the idea. A vast amount of kinetic data on families of substrates and related competitive inhibitors support the idea and numerous X-ray structures of enzymes with bound inhibitors or with very slow substrates have given visual evidence of the reality of the lock-and-key concept. Directed mutation of genes of many enzymes of known three-dimensional structure has provided additional proof. 

Using directed mutation of the cloned receptor gene, Lys 1018 in the ATP-binding part of the tyrosine kinase domain was replaced by alanine. This caused a loss both of kinase activity and of biologic response to insulin.362 Thus, both the tyrosine kinase activity and autophosphorylation appear essential. If so, aggregation of two or more receptors may increase the extent of autophosphorylation and initiate a response. 

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