Side-directed mutagenesis

While the production of D-amino acids is well established the preparation of L-amino acids is difficult due to the limited selectivity and narrow substrate spectrum of L-hydantoinases. This can be circumvented by employing rather un-selective hydantoinases in combination with very enantioselective L-carbamoyl-ases and carbamoyl racemases [90]. Furthermore, a D-hydantoinase has been genetically modified and converted into a L-hydantoinase. This enzyme can be used on a 100-kg scale for the production of L-tert-leucine [34]. Finally, the fact that the X-ray structure of an L-hydantoinase is known gives hope that side-directed mutagenesis will lead to improved L-hydantoinases [91]. 

Other pre-requisites for crystallization are good stability and sufficient solubility (typically 5-15mg/ml, but some highly soluble proteins may require a much higher concentration, while others can be crystallized at a concentration between 1 and 5mg/ml). Solubility and stability can sometimes be improved by side-directed mutagenesis. Both random mutagenesis approaches and rational ones have been proposed and successfully used. Alternatively, one may consider using another species instead of the human protein. 

Moreover, technological applications can take advant e irom recent advance in PSII molecular biology and in site-directed mut enesis, which has produced a number of mutants of bacteria, algae and cyanobacteria (e.g., Rhodobacter, Synechocystis, Chlantydomonai) resistant to extreme conditions. The mutants show altered aminoacid composition of the D1 protein. Cysteines and Histidines, which can help immobiUsation of the proteins, have also been introduced into the D1 protein by side-directed mutagenesis. ... 

Residue 189 is at the bottom of the specificity pocket. In trypsin the Asp residue at this position interacts with the positively charged side chains Lys or Arg of a substrate. This accounts for the preference of trypsin to cleave adjacent to these residues. In chymotrypsin there is a Ser residue at position 189, which does not interfere with the binding of the substrate. Bulky aromatic groups are therefore preferred by chymotrypsin since such side chains fill up the mainly hydrophobic specificity pocket. It has now become clear, however, from site-directed mutagenesis experiments that this simple picture does not tell the whole story. 

The contributions of hydrogen bond donors to catalysis can be estimated by site-directed mutagenesis studies in cases where the hydrogen bond donor is located in the amino acid side chain. Deletion of the main chain NH is only possible by substituting the amino acid with a proUne. In all cases, the effects of the substitution to key enzyme kinetic parameters, and K, should be checked. Typically, the oxyanion hole residues contribute only Uttle to the binding of substrate [19-21]. This is reflected in the values, which typically remain very similar... 

Use can be made of the affinities of metals to imidazoles to modify enzyme activity. For example, site-directed mutagenesis was used to add a second histidine to a serine protease in order to enhance the interaction between a transition metal ion and the side chain of His-57 (Higaki et al, 1990). The strengths of association of metals were found to be Cu (21 yM) > Ni (49 /xM) > (128 p.M). This is the order of associa-... 

In order to increase the understanding of ThDP-dependent enzymes, the identification of amino acid side chains important for the catalysis of the carboligase reaction in pyruvate decarboxylase from Zymomonas mohilis (E.C. 4.1.1.1) and benzoylformate decarboxylase from Pseudomonasputida (E.C. 4.1.1.7) was a major task. Using site-directed mutagenesis and directed evolution, various enzyme variants were obtained, differing in substrate specificity and enantioselectivity. 

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