Enzymes evolutionary strategy

The utilization of evolutionary strategies in the laboratory can be illustrated with proteins that catalyze simple metabolic reactions. One of the simplest such reactions is the conversion of chorismate to prephenate (Fig. 3.3), a [3,3]-sigmatropic rearrangement. This transformation is a key step in the shikimate pathway leading to aromatic amino acids in plants and lower organisms [28, 29]. It is accelerated more than a million-fold by enzymes called chorismate mutases [30],... 

Nature has solved the problem of protein design through the mechanism of Darwinian evolution. Every one of the proteins in our cells, from enzymes and receptors to structural proteins, has arisen by this process. As the examples presented here with chor-ismate mutase amply demonstrate, evolutionary strategies can also be successfully exploited in the laboratory to study the structure and function of existing proteins and to engineer new ones. 

Naturally, the first step of each evolutionary project is the creation of diversity. The most straightforward approach to create a library of proteins is to introduce random mutations into the gene of interest by techniques such as error-prone PCR or saturation mutagenesis. The success of random mutagenesis strategies is witnessed by their ample appearances in the different chapters of this book describing case studies of particular classes of proteins and enzymes. In addition, recombination of mutant... 

The catalytic strategy that an enzyme develops over evolutionary time is dictated by the chemistry of the reaction being catalyzed. The prolyl isomerases that have been studied to date are able to simply stabilize the nonenzymatic transition state without formation of covalent intermediates. Based on a value of lO" sec for CyP (Harrison and Stein, 1992 Kofron et ai, 1991) and a of sec" for the cis-to-trans isomerization of Suc-Ala-Ala-cis-Pro-Phe-pNA, we calculate an acceleration factor, of 10 , which corresponds to a transition state... 

Recently, a controversial debate has arisen about whether the optimization of enzyme catalysis may entail the evolutionary implementation of chemical strategies that increase the probability of tunneling and thereby accelerate reaction rates [7]. Kinetic isotope effect experiments have indicated that hydrogen tunneling plays an important role in many proton and hydride transfer reactions in enzymes [8, 9]. Enzyme catalysis of horse liver alcohol dehydrogenase may be understood by a model of vibrationally enhanced proton transfer tunneling [10]. Furthermore, the double proton transfer reaction in DNA base pairs has been studied in detail and even been hypothesized as a possible source of spontaneous mutation [11-13]. 

Evolutionary pressures have constrained pertussis toxin to a mechanism with significant nucleophile participation while RTA has evolved a catalytic strategy with an oxocarbenium ion intermediate that reacts quickly with nearby water molecules. By this logic, other ADP-ribosylating toxins, such as cholera and diphtheria toxins, would be expected to proceed through relatively synchronous transition states. Nucleoside hydrolases (enzymes that hydrolyze nucleosides to ribose and a purine or pyrimidine base) could use mechanisms with oxocarbenium ion intermediates, though the transition states characterized to date have been A Dn (see below). 

Arguments that invoke evolutionary forces are distinct, of course, from questions of how different catalytic strategies are effected through specific enzyme-substrate interactions. Because very detailed pictures of transition states have not previously been available, these kinds of questions are only now beginning to be addressed. 

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