Enzyme catalyzed evolution

The patterns of autocatalysis with respect to parabolic and exponential reaction courses, which strongly affect the conclusions of Eigen s evolution experiments concerning the decision criteria for mutant selection and coexistence [5 b, 40 h, k], can now be derived from the thermodynamic data of the matrix patterns and their reactivities, and offer quite new views, with autocatalytic cooperation between competitive species [40 k]. Separate from enzyme-catalyzed evolution experiments with RNA and DNA systems, basic questions of prebiotic behavior can for the first time become the subject of detailed experimental research [40 k, 43]. While continuing their studies on more complex autocatalysis patterns, von Kie-drowski et al. diagnosed modulation of molecular recognition as an operational deficit of earlier artificial self-replicational nucleic acid systems with regard to exponential reaction courses, and identified it as an ideal aim for future models [44]. On its way to the nucleoprotein system, evolution must have... 

Since most synthetic applications require enzymes catalyzing nonnatural substrates, their properties often have to be improved. One way to achieve this is to optimize reaction conditions such as pH, temperature, solvents, additives, etc. [6-9]. Another way is to modulate the substrates without compromising the synthetic efficiency of the overall reaction [10]. In most cases for commercial manufacturing, however, the protein sequences have to be altered to enhance reactivity, stereoselectivity and stability. It was estimated that over 30 commercial enzymes worldwide have been engineered for industrial applications [11]. Precise prediction of which amino acids to mutate is difficult to achieve. Since the mid 1990s, directed evolution... 

Williams, G.J., Domann, S., Nelson, A. and Berry, A. (2003) Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution. Proceedings of the National Academy of Sciences of the United States of America, 100, 3143-3148. 

Life is sustained by a complex web of chemical reactions. Catalysts, molecules that accelerate the rate of a chemical reaction but that are unchanged by the overall reaction, are essential for life as most reactions would otherwise occur far too slowly. Indeed, it can be argued that the evolution of life is essentially the story of the evolution of catalysis. In nature, most catalysts are proteins and these catalytic proteins, or enzymes, are one of the most remarkable classes of molecules to have been generated during evolution. Enzymes catalyze an enormous range of different reactions and their performances typically far exceed those of man-made catalysts. They can accelerate reactions by anything up to 10 -fold relative to the uncatalyzed reaction, enabling reactions that would otherwise have half-lives of tens of millions of years to be performed in milliseconds. 

The two-component enzyme catalyzes the reduction of N2 to 2NH4+ or of C2H2 to (exclusively) C2H4 and the evolution of H2 with electrons supplied by the reductants named above. ATP... 

Although enzyme-catalyzed reactions are described in many other entries in this Handbook, some mention of the time-evolution of an enzymatic process should be considered here. Shown in Fig. 10 is an representation of a typical reaction progress curve. A rapid rise in the concentration of reactant-bound species ES + +... 

A quantitative expression developed by Albery and Knowles to describe the effectiveness of a catalyst in accelerating a chemical reaction. The function, which depends on magnitude of the rate constants describing individual steps in the reaction, reaches a limiting value of unity when the reaction rate is controlled by diffusion. For the interconversion of dihydroxacetone phosphate and glyceraldehyde 3-phosphate, the efficiency function equals 2.5 x 10 for a simple carboxylate catalyst in a nonenzymic process and 0.6 for the enzyme-catalyzed process. Albery and Knowles suggest that evolution has produced a nearly perfect catalyst in the form of triose-phosphate isomerase. See Reaction Coordinate Diagram... 

Burbaum et al. considered how kinetic/thermodynamic features of present-day enzyme-catalyzed reactions suggest that enzyme evolution tends to maximize catalytic effectiveness. They analyzed Uni Uni enzymes in terms of reaction energetics. Catalytically optimized enzymes... 

An advantage of these enzymes is that they are stereocomplementary, in that they can synthesize the four possible diastereoisomers of vicinal diols from achiral aldehyde acceptors and DHAP (Scheme 4.2). Although this statement is generally used and accepted, it is not completely true since tagatose-l,6-bisphosphate aldolase (TBPA) from Escherichia coli-the only TBPA that has been investigated in terms of its use in synthesis-does not seems to control the stereochemistry of the aldol reaction when aldehydes different from the natural substrate were used as acceptors [7]. However, this situation could be modified soon since it has been demonstrated that the stereochemical course of TBPA-catalyzed C—C bond formation may be modified by enzyme-directed evolution [8]. 

Spectrophotometric assays can be used for the estimation of the enantiosel-ectivity of enzymatic reactions. Reetz and coworkers tested 48 mutants of a lipase produced by epPCR on a standard 96-well microtiter plate by incubating them in parallel with the pure R- and S-configured enantiomers of the substrate (R/S-4-nitrophenol esters) [10]. The proceeding of the enzyme catalyzed cleavage of the ester substrate was followed by UV absorption at 410 nm. Both reaction rates are then compared to estimate the enantiomeric excess (ee-value). They tested 1000 mutants in a first run, selecting 12 of them for development of a second generation. In this way they were able to increase the enantiomeric excess from 2% for the first mutants to 88% after four rounds of evolutive optimization. 

Metalloenzymes or metal ion-activated enzymes catalyze an enormous variety of organic reactions that are not restricted to any particular reaction class, but appear as catalysts for all types of reactions. Thus neither the presence of the metal ion nor the reaction type seems to be restrictive as far as metal-assisted enzyme catalysis is concerned. In some cases the metal ion appears to function as an electron acceptor or donor, but flavin cofactors have substituted as redox centers during evolution in some enzymes. 

As already demonstrated in Ch. 11, Section 11.1.3 (Figure 11.2), many different enzymes can evolve from a common protein structure. The enzymes in Figure 11.2 feature a highly conserved ajffbarrel structure, but neither the amino acid sequence nor the function of these proteins is constant over the timescale of evolution. Conversely, as mentioned in Section 11.1.3 as well, an enzyme catalyzing a certain activity can otherwise feature very different properties in different organ-... 

Zha, D., Wilensek, S., Hermes, M. Jaeger, K. E. Reetz, M. T. Complete reversal of enantioselectivity of an enzyme-catalyzed reaction by directed evolution. Chem. Commun. 2001,2664-2665. 

Enzyme-catalyzed reactions, which are characteristically reversible under physiologic conditions, are ideally suited to the generation of dynamic combinatorial libraries. Many enzymes with broad specificity (required for library diversity) are already commercially available, and the application of modem techniques in directed evolution may be expected to increase their number. 

Zha D, Wilensek S, Hermes M, Jaeger K-E, Reetz MT (2001) Complete reversal of enantioselectivity of an enzyme-catalyzed reaction by directed evolution. Chem Commun (Cambridge UK) 2664-2665 Zou JY, Hallberg BM, Bergfors T, Oesch F, Arand M, Mowbray SL, Jones TA (2000) Structure of Aspergillus niger epoxide hydrolase at 1.8 resolution Implications for the structure and function of the mammalian microsomal class of epoxide hydrolases. Structure (London) 8 111-122... 

Williams GJ, Domann S, Nelson A, Berry A. Modifying die stereochemistry of an enzyme-catalyzed reaction by directed evolution. Proc. Natl. Acad. Sci. U.S.A. 2003 100 3143-3148. 

---