On the failure of de novo-designed peptides as biocatalysts.

Abstract

While the elegance and efficiency of enzymatic catalysis have long tempted chemists and biochemists with reductionist leanings to try to mimic the functions of natural enzymes in much smaller peptides, such efforts have only rarely produced catalysts with biologically interesting properties. However, the advent of genetic engineering and hybridoma technology and the discovery ofcatalytic RNA have led to new and very promising alternative means of biocatalyst development. Synthetic chemists have also had some success in creating nonpeptide catalysts with certain enzyme-like characteristics, although their rates and specificities are generally much poorer than those exhibited by the best novel biocatalysts based on natural structures. A comparison of the various approaches from theoretical and practical viewpoints is presented. It is suggested that, given our current level of understanding, the most fruitful methods may incorporate both iterative selection strategies and rationally chosen small perturbations, superimposed on frameworks designed by nature.A thorough understanding of the chemical and structural bases of biological catalysis would lead to advances in medicine, synthetic chemistry, materials science, agriculture, and other fields. Such a level of insight, when it exists, will likely be signaled by a clear demonstration of the ability to construct, from first principles, a range of catalysts capable of transmuting both biopolymers and small molecules to desired products with high specificity and acceptable efficiency. The specialized proteins known as enzymes are the molecules that usually fill this role in nature, and the most likely means by which we could achieve the objective of catalysis to specification is by attaining an understanding of enzyme structure and function sufficient to allow design and construction of new molecules based on the same principles. This degree of understanding has been elusive, and there is as yet no case of an protein or peptide designed de novo that catalyzes a reaction of biological interest with efficiency and specificity comparable to that of natural enzymes or a novel reaction not carried out by enzymes. However, competing strategies of developing novel biocatalysts which make use of the frameworks of natural proteins have enjoyed considerable success in catalyzing both transformations analogous to the cognate reactions of natural enzymes and, in some cases, entirely novel reactions. The two major techniques in this category are catalytic antibodies (refs. 1 and 2; for a recent review, see ref.3) and reengineered natural enzymes (RNEs; for reviews, see refs. 4 and 5). Catalytic antibodies, which were originally reported in 1986, can be characterized as antibodies directed against haptens, which are usually synthetic analogs of the transition states of the chemical reactions to be catalyzed. Reengineering of enzymes has been possible in principle since the advent of genetic engineering, more than two decades ago; a review of Medline entries indicated that st...

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“…After cleavage from the resin by trifluoroacetic acid, the crude peptide obtained was purified by HPLC chromatography with an ODS column, 20 ϫ 250 mm, with a gradient system of water/acetonitrile containing 0.1% trifluoroacetic acid. Amino acid analysis was performed after hydrolysis in 5.7 M HCl in a sealed tube at 110°C for 24 h. Analytical data obtained were as follows: Gly, 6.2 (6); Ala, 9.5 (10); Leu, 26.5 (25); Asp, 3.0 (3); Pro, 2.9 (3); Tyr, 3.1 (3); Lys, 18.9 (18). Molecular weight was determined by fast atom bombardment mass spectroscopy using a JEOL JMX-HX100: base peak, 7555.1; calculated for C367H639O77N91⅐H ϩ , 7554.8.…”

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