I= PRODUCTION AND USE OF TAILOR-MADE BIOLOGICAL CATALYSTS
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Title of Thesis
PRODUCTION AND USE OF TAILOR-MADE BIOLOGICAL CATALYSTS

Author(s)
Mufti Asif Ullah
Institute/University/Department Details
University of Karachi/ H.E.J. Research Institute of Chemistry
Session
1999
Subject
Chemistry
Number of Pages
113
Keywords (Extracted from title, table of contents and abstract of thesis)
biological catalysts, enzymes, chorismate mutase, tailored biocatalysts, monoclonal antibodies, dna manipulations, diels alser reaction

Abstract
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 5.4.99.5) 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.

Download Full Thesis
1327.12 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
86.61 KB
2 1 Introduction to Tailored Biocatalysts 1
249.7 KB
  1.1 Site-directed mutagenesis and genetic selection in vivo 4
  1.2 Fruitful enzyme reengineering and selection 7
  1.3 Claisen rearrangement catalyzed by chorismate mutase 9
  1.4 Monoclonal antibodies: a powerful tool of catalysis 16
  1.5 Catalytic antibodies 19
  1.6 The antibody catalysis of diels alder reaction 27
  1.7 Aims of research 30
3 2 Experimental 32
207.61 KB
  2.1 Section-A Study of the active site of chorismate mutase by combinatorial mutagenesis And selection 32
  2.2 Section-B Purification, characterization and activation Parameters of Bacillus Subtillis Chorismate mutase 38
  2.3 Section-C Towards the antibody catalysis of diels alder reaction 14
4 3 Results and Discussion 58
462.21 KB
  3.1 Novel genetic selection strategy in vivo 58
  3.2 Construction of partially randomized aroH libraries 62
  3.3 Alternative active site residues founded by selection 66
  3.4 Characterization of a subset of selected active site variants 77
  3.5 Chorismate mutas purification 80
  3.6 Kinetic characterization of claisen rearrangement 85
  3.7 Electrostatic catalysis of the claisen rearrangement 86
  3.8 Determination of activation parameters: evidence for an unfavorable Entropy ( S) 90
  3.9 Hapten design and synthesis for diels alder reaction 94
  3.10 Catalytic screening of antibodies for diels alder reaction 97
5 4 References 101
69.14 KB
6 5 List of Figures 109
17.68 KB
7 6 List of Tables 111
8.55 KB
8 7 List of abbreviations 112
393.19 KB