Mutation site-directed

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...

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)... 

M. R. Lentz, R. G. Webster, and G. M. Air, Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism, Biochemistry, 26 (1987) 5351-5358. 

However, there are also examples of prenyltransferase in which substrates are only poorly accepted with low rate constants (Kc.lt/Knl). especially when their structures differ significantly from the natural substrates of the enzymes. There are two ways to avoid such drawbacks in future (1) enzyme optimisation by site-directed mutation and (2) significantly increased catalyst overexpression, so that the amount of enzyme is not the limiting factor. 

Usanov SA, Graham SE, Lepesheva GI, et al. Probing the interaction of bovine cytochrome P450scc (CYP11A1) with adrenodoxin evaluating site-directed mutations by molecular modeling. Biochemistry 2002 41 8310-8320. 

Malmberg A, Strange PG. Site-directed mutations in the third intracellular loop of the serotonin 5-HT1A receptor alter G protein coupling from Gj to Gs in a ligand-dependent manner. J Neurochem 2000 75 1283-1293. 

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