Enzymes directed mutations

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. ... 

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. 

Eklund et al. suggested that the side chains of Ser 48 and His 51 act as a proton relay system to remove the proton from the alcohol, in step b of Eq. 15-7, leaving the transient zinc-bound alcoholate ion, which can then transfer a hydride ion to NAD+, in step c.52 The shaded hydrogen atom leaves as H+. The role of His 51 as a base is supported by studies of the inactivation of the horse liver enzyme by diethyl pyrocarbonate57 and by directed mutation of yeast and liver enzymes. When His 51 was substituted by Gin the pKa of 7 was abolished and the activity was decreased ten-fold.58... 

Formation of dUMP in eukaryotes may occur by hydrolytic removal of phosphate from dUDP or from the conversions dCDP —> dCMP —> dUMP (steps k and V, Fig. 25-14). A more roundabout pathway is employed by E. coli dCDP —> dCTP —> dUTP —> dUMP (steps /c, /, and m, Fig. 25-14). One of the intermediates is dUTP. DNA polymerases are able to incorporate dUMP from this compound into polynucleotides to form uracil-containing DNA. Tire only reason that this does not happen extensively within cells is that dUTP is rapidly converted to dUMP by a pyrophosphatase (step m, Fig. 25-14). Tire uracil that is incorporated into DNA is later removed by a repair enzyme (Chapter 27). The presence of dUTP in DNA provides the basis for one of the most widely used methods of directed mutation of DNA (Chapter 26). 

Enzyme or DNA mutations may be likened to the fluctuations of a surface. In principle, therefore, in developing upon this work of Ya.B., one might consider a natural, physical explanation of directed mutations and of the influence of the environment (an adsorbate entering a reaction) on the mutations. 

Observed differences between strains of rats and mice, as described below, may be the result of gene polymorphisms. In cases involving insecticide selection pressure, resistant populations may arise as a result of direct mutations of insecticide-metabolizing enzymes and/or insecticide target sites that are passed on to succeeding generations. 

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. 

These investigations also showed that the conversion of ECB to ECB nucleus would proceed more rapidly if ECB were first solubilized in a suitable solvent such as methanol or acetone. However, if the concentration of solvent was too high, the enzyme activity was reduced. Ideally, the enzyme itself could be tailored to suit the industrially preferred conditions (e.g., to make it more resistant to solvent or active at a different pH). One method for achieving this is to use directed evolution [42], whereby genes encoding the enzyme are mutated, screened and then recombined in vitro. Although the contributions of individual amino acid mutations are small, the accumulation of multiple mutations by directed evolution allows significant improvement in the biocatalyst for reactions on substrates or under conditions not already optimized in nature. This approach was used by Arnold and Moore [43] to make a 150-fold improvement in the activity of a -nitrobenzyl esterase in the presence of 15% DMSO. 

Our new ability to obtain a highly purified preparation of a bacterial coupling factor PPase, in combination with our recently obtained capacity to clone any part of the R. rubrum genome, may open the road to a detailed study of the molecular properties of the enzyme. The techniques now available for directed mutations of bacterial genomes may give us possibilities to alter the dynamic properties of the enzyme in ways that should provide added insight into the mechanism of formation and utilization of inorganic pyrophosphate. 

Sebastian S, Wilson JE, Muhchak A, Garavito RM. Allosteric regulation of type I hexokinase A site-directed mutational study indicating location of the functional glucose 6-phosphate binding site in the N-terminal half of the enzyme. Arch Biochem Biophys 1999 362 203-10. 

The examples in this section have been chosen to provide an in-depth presentation showing how RSSF currently has been applied to the study of biological systems. These applications include the study of isotope effects on enzyme-catalyzed reactions, the investigation of substrate-metal ion interactions in metalloenzymes, the search for and identification of covalent intermediates in enzyme-catalyzed processes, the analysis of the effects of site-directed mutations on enzyme catalytic mechanism, and the exploitation of natural and artificial chromophores as probes of allosteric processes. 

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