Enzyme catalysts mutation

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

It is well understood that the synthesis and modihcation of metabolites is under enzymatic control. The enzymes may function as catalysts, and the reactions themselves are not restricted only to living systems. So the evolution of biosynthetic capacity is largely the result of changes in enzymes by mutation, gene duplication, and other familiar processes. The organisms synthesize and modify secondary metabolites in a stepwise fashion, much as organic chemists do, and in neither case are the laws of nature violated. 

Benning, M.M., Taylor, K.L., Liu, R.-Q., Yang, G., Xiang, H., Wesenberg, G., Dunaway-Mariano, D. Holden, H.M. (1996) Biochemistry, 35, 8103-8109, Stmcture of 4-chlorobenzoyl coenzyme A dehalogenase determined to 1.8 A resolution an enzyme catalyst generated via adaptive mutation. 

These examples are part of a broader design scheme to combine catalytic metal complexes with a protein as chiral scaffold to obtain a hybrid catalyst combining the catalytic potential of the metal complex with the enantioselectivity and evolvability of the protein host [11]. One of the first examples of such systems combined a biotinylated rhodium complex with avidin to obtain an enantioselective hydrogenation catalyst [28]. Most significantly, it has been shovm that mutation-based improvements of enantioselectivity are possible in these hybrid catalysts as for enzymes (Figure 3.7) [29]. 

In some cases, mutation can lead to enhanced catalytic ability of the enzyme. Results for the mutation Thr-51 to Pro-51 (Wilkinson et al 1984) have been mentioned previously. The results for this and for the mutation Thr-51 to Ala-51 (Fersht e/ al., 1985) are also shown in Table 18. These mutations and that of Thr-51 to Cys-51 have been studied in some detail (Ho and Fersht, 1986). In each case it is found that the transition state is stabilized for formation of tyrosine adenylate from tyrosine and ATP within the enzyme the mutant Thr-51 to Pro-51 increases the rate coefficient for the reaction by a factor of 20. However, the enzyme-bound tyrosine adenylate is also stabilized by the mutation and this results in a reduced rate of reaction of tyrosine adenylate with tRNA (48), the second step in the process catalysed by tyrosine tRNA synthetase. Overall, therefore, the mutants are poorer catalysts for the formation of aminoacyl tRNA. The enzyme from E. coli has the residue Pro-51 whereas Thr-51 is present in the enzyme from B. stearothermophilus. The enzyme from E. coli is more active than the latter enzyme in both the formation of tyrosine adenylate and in the aminoacyla-tion of tRNA (Jones et al., 1986b). It is therefore suggested (Ho and Fersht, 1986) that the enzyme from E. coli with Pro-51 must additionally have evolved ways of stabilizing the transition state for formation of tyrosine adenylate without the concomitant stabilization of tyrosine adenylate and reduction in the rate of aminoacylation of tRNA found for the Pro-51 mutant. 

In the last Section 6.4 new supramolecular approaches to construct synthetic biohybrid catalysts are described. So-called giant amphiphiles composed of a (hydrophilic) enzyme headgroup and a synthetic apolar tail have been prepared. These biohybrid amphiphilic compounds self-assemble in water to yield enzyme fibers and enzyme reaction vessels, which have been studied with respect to their catalytic properties. As part of this project, catalytic studies on single enzyme molecules have also been carried out, providing information on how enzymes really work. These latter studies have the potential to allow us to investigate in precise detail how slight modifications ofthe enzyme, e.g., by attaching a polymer tail, or a specific mutation, actually infiuence the catalytic activity. 

The design of supramolecular catalysts may make use of biological materials and processes for tailoring appropriate recognition sites and achieving high rates and selectivities of reactions. Modified enzymes obtained by chemical mutation [5.70] or by protein engineering [5.71] represent biochemical approaches to artificial catalysts. 

Besides these rather complex coenzyme-dependent enzymes, the none-coenzyme requiring protease subtilisin is the most extensively mutated enzyme. The substrate specificity of the enzyme as well as its dependence on pH and its stability were altered by site-directed mutagenesis [72-78]. As the knowledge about exact details of the structure and active site of the enzyme is essential for the application of this method, progress in this field is difficult to achieve. Site-directed mutagenesis as a means of catalyst improvements will be used only after extensive application of conventional optimization procedures. 

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 results were unprecedented in the catalytic antibody area. Traditionally, antibody catalysts are very specific and only catalyze the reaction of a single substrate (or substrate combination). This is because in the normal case of immunization, a series of somatic mutations usually leads to an increase in binding specihcity toward the inducing antigen. Antibody catalysts generated from this process have the restricted substrate specihcity characteristic of most natural enzymes. 

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