Amino acid substitution enzyme enzymes

As delineated above, if the mutation rate is doubled in order to induce an average of two amino acid substitutions per enzyme molecule, about 15 million different mutants are theoretically possible. A simple calculation shows that if 3000 mutants are then screened, less than 0.02 % of the total protein sequence space would be scanned. In the case of three amino acid exchanges, the statistics are even more drastic. Nevertheless, experiments at relatively high mutation rate leading to an average of 2 - 3 amino acids substitutions were performed. About 15 000 variants of the initial library were screened for enantioselectivity in the model reaction. Several (S)- and even (R)-... 

Dl. Daar, I. O., Artymiuk, P. J., Phillips, D. C and Maquat, L. E., Human triosephosphate iso-merase deficiency. A single amino acid substitution results in a thermolabile enzyme. Proc. Natl. Acad. Sci. U.S.A. 83,7903-7907 (1986). 

F5. Fujii, H., Krietsch, W. K. G., and Yoshida, A., A single amino acid substitution (Asp -> Asn) in a phosphoglycerate kinase variant (PGK Miinchen) associated with enzyme deficiency. J. Biol. Chem. 255,6421-6423 (1980). 

LHRH (see Fig. 6.16) is highly sensitive to proteolytic degradation. Enzymes with particular activity toward LHRH include angiotensin-converting enzyme, neprilysin, and thimet oligopeptidase (see Sect. 6.4.2 and Table 6.6). D-Amino acid substitution in position 6, 7, or 10 has led to the de-... 

Amino Acid Substitution The naturally occurring or experimentally induced replacement of one or more amino acids in a protein with another. If a functionally equivalent amino add is substituted, the protein may retain wild-type activity. Substitution may also diminish or eliminate protein function. Experimentally induced substitution is often used to study enzyme activities and binding site properties. [NIH]... 

This chapter aims to summarize our efforts to investigate the effects of fluorinated amino acid substitutes on the interactions with natural protein environments. In addition to a rather specific example concerning the interactions of small peptides with a proteolytic enzyme, we present a simple polypeptide model that aids for a systematic investigation of the interaction pattern of amino acids that differ in side chain length as well as fluorine content within both a hydrophobic and hydrophilic protein environment. Amino acid side chain fluoiination highly affects polypeptide folding due to steric effects, polarization, and fluorous interactions. 

We now take account of the specific character of the incorporation of these a-substituted amino acids into polypeptides. Enzyme inhibition by a-fluoromethyl amino acids is detailed in Chapter 7. 

Synthesis of unusual amino acids, peptidomimetics, synthetic enzymes, and new drugs containing (5-lactam rings as an integral part of their structure, has currently renewed interest worldwide in this four-membered heterocycle [52-54]. Furthermore, the appropriately substituted and configured(5-lactam frameworks now constitute effective tools for the incorporation of a wide variety of both (5- and a-amino acids in short peptide segments of various biologically active molecules. 

Branched-chain aminotransferases (BCATs) evolved from aspartate aminotransferases (AATs) showed a record 105- to 2 x 106-fold improvement in catalytic efficiency (kcat/KM). Not only were the 13-17 amino acid substitutions concentrated in the most active mutants, but all but one mutated amino acid residues are located far from the active site. With directed evolution, enantioselectivities can be improved on enantiounspecific enzymes (from E = 1.1 to 25.8) and even inverted to yield the opposite enantiomer in comparison to the wild type (40% d- to both 90% d- and 20% L-). 

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