Asif Ullah, Mufti (1999) PRODUCTION AND USE OF TAILOR-MADE BIOLOGICAL CATALYSTS. PhD thesis, University of Karachi, Karachi.
The biosynthetic evolution of enzymes has taken over millions of years. These natural catalysts make the complicated process of life possible and have also become extremely viable tools in basic research, medicine and industry. There are a number of useful chemical reactions which can be catalyzed by enzymes in the laboratory, however there are still a large number of chemical transformation for which no suitable enzyme is known. My doctoral research has been concerned with developing strategies to generate and characterize catalysts, either by tailoring the active site of the enzyme or by raising catalytic monoclonal antibodies against transition state analogs. My work has been built on previous achievements in this area, focusing on the investigations of factors important in protein engineering (tailoring active sites of enzyes) and antibody catalysis. We have tailored the active site of chorismate mutase (EC 188.8.131.52) which catalyzes the interamolecular rearrangement of chorismate to prephenate. Glu78 and Arg90 in the active site of the chorismate mutase enzyme from Bacillus subtillis was in close proximity to the substrate and may contribute to efficient catalysis by electrostatically stabilizing the presumed dipolar transition state that would result upon scission of the C-O bond which substantially precedes C-C bond formation. To test this idea we have developed a novel complementation system for chorismate mutase activity in Eshcerichia coli by reengineering parts of the aromatic amino acid biosynthetic pathway. The condons for Glu78 and Arg90 were randomized alone and in combination with Cys78 and Cys88, and active clones were selected. Our results support the nation that an electrostatic gradient in the active site is a major factor in chorismate mutase catalysis. The new selection system, in conjugation with combinatorial mutagenesis, renders the mechanism of the natural enzyme(s) accessible to further exploration and opens avenues for the improvement of the first generation of catalytic antibodies with chorismate mutase activity. We have characterized chorismate mutase by determining the activation parameters. Chorismate mutase is known to accelerate the chorismate to prephenate rearrangement, in part by significantly lowering the entropy barrier for the reaction. We have found that S(-9.1 1.2 ev) is nearly as unfavorable as the activation entropy for the uncatalyzed process. Our results suggest that chorismate mutase catalysts show greater mechanistic versatility than commonly believed. Research towards expanding the reaction repertoire of catalytic antibodies has involved designing and synthesizing transition state analogs for the Diels Alser reaction.
|Item Type:||Thesis (PhD)|
|Uncontrolled Keywords:||biological catalysts, enzymes, chorismate mutase, tailored biocatalysts, monoclonal antibodies, dna manipulations, diels alser reaction|
|Subjects:||Physical Sciences (f) > Chemistry(f2)|
|Deposited By:||Mr. Muhammad Asif|
|Deposited On:||08 Nov 2006|
|Last Modified:||04 Oct 2007 21:03|
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