Site-directed mutagenesis enzyme modification

The following new trends in enzymatic synthesis can be delineated the development of new enzymatic reactions enzyme immobilization and stabilization the use of organic solvents and two phase systems site-directed mutagenesis chemical modification of enzymes antibody catalysis catalysis by RNA and DNA de novo design ofbiocatalists employment of recombinant DNA for production of enzymes and use computational and combinatorial methods... 

Before the introduction of site-directed mutagenesis, chemical modification was the predominant means of altering a specific amino acid the earliest report with lysozyme was from Parsons and Raftery (241), who prepared the ethyl ester derivative of Asp-52 by reaction with triethyloxonium fluoroborate the modified enzyme lost catalytic function but not substrate affinity. An ethyleneimine reaction product of Asp-52 has also been prepared with similar effects on catalysis (242). Sharon and co-workers modified and then regenerated Asp-52 to eliminate concern that inactivation results from experimental manipulation rather than specific amino acid modification (243, 244). Thus, Asp-52 was first esteri-fied with an epoxypropyl-jS-glycoside derivative of di-(/V-acetyl-D-glucosamine), then reduced to homoserine or hydrolyzed to return the free aspartate. Both the... 

Groen, H., Bech, L.M., Branner, S. andBreddam, K. (1990) A highly active and oxidation-resistant mutant subtilisin-like enzyme produced by a combination of site-directed mutagenesis and chentical modification. Eur. J. Biochem., 194, 897-901. 

On the basis of chemical modification studies, Tyr 198 of carboxypeptidase A was proposed to act as a proton donor (i.e., a general acid) in the mechanism of catalysis. However, when Tyr 198 was replaced with Phe by means of site-directed mutagenesis, the modified enzyme retained substantial enzymatic activity, indicating that the tyrosyl hydroxyl may not have a specific role in catalysis. 

The principle advantage of the physical labeling method is the possibility of receiving direct information about the structure, mobility and local micropolarity of certain parts of a molecular object of any molecular mass. Developments in synthetic chemistry, biochemistry and site-directed mutagenesis have provided researchers with a wide assortment of labels and probes, and have paved the way for the specific modification of protein function groups, including enzyme active sites. 

Thus far, the only a-1,4 glucan synthase reported to be overexpressed with high activity is the E. coli enzyme. This system should be further exploited with respect to the methodologies of chemical modification, site-directed mutagenesis, and attempts to determine its three-dimensional structure. 

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