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Dissertation Tony E RATIONAL DESIGN OF ORGANOPHOSPHORUS HYDROLASE FOR THE DEGRADATION AND DETECTION OF NEUROTOXIC PESTICIDES AND CHEMICAL WARFARE AGENTS A Dissertation by TONY ELVERN REEVES Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2006 Major Subject: Biochemistry RATIONAL DESIGN OF ORGANOPHOSPHORUS HYDROLASE FOR THE DEGRADATION AND DETECTION OF NEUROTOXIC PESTICIDES AND CHEMICAL WARFARE AGENTS A Dissertation by TONY ELVERN REEVES Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, James Wild Committee Members, Evelyn Castiglioni Janet Grimsley Paul Lindahl Martin Scholtz Head of Department, Greg Reinhart May 2006 Major Subject: Biochemistry iii ABSTRACT Rational Design of Organophosphorus Hydrolase for the Degradation and Detection of Neurotoxic Pesticides and Chemical Warfare Agents. (May 2006) Tony Elvern Reeves, B.S.; M.S., Tarleton State University Chair of Advisory Committee: Dr. James R. Wild It is critical to consider the balance between the catalytic capabilities of an enzyme and the inherent structural stability of the protein when developing enzymes for specific applications. Rational site directed mutagenesis has been used to explore the role of residues 254 and 257 in the global stability and catalytic specificities of organophosphorus hydrolase (OPH, EC 3.1.8.1). Substitution of residues H254 and H257, which are located near the active site, had a marked effect on both the global stability and substrate specificity of the enzyme. For example, the ΔGT for the double mutation Co2+ H254R H257L (RL) enzyme variant was 19.6 kcal/mol, 5.7 kcal/mol less than that of the wild type enzyme. At the same time, the altered enzyme was catalytically more effective against VX and VR (Russian VX), as compared to the wild type enzyme. Limited proteolysis verified the importance of residues 254 and 257 for functional stability, evidenced by enhanced resistance to irreversible unfolding associated with thermal denaturation. It has been possible to construct third generation OPH variants, which are more stable than the wild type enzyme, with a 10 °C increase in the apparent melting temperature (TM app), yet retained desirable catalytic properties. It appeared that aromatic stacking and cation-π interactions involving near active site iv residues not only affected activity but significantly contributed to the chemical and thermal stability of OPH. Rational design was used to develop an enzyme with an optimized orientation on a catalytically active biosensor surface. In these studies, lysine side chains located on the surface of OPH were used to create attachment sites to a surface plasmon resonance sensor resulting in an ensemble of enzyme orientations. Some of these orientations could be functionally restrictive if the active site is oriented toward the sensor surface. Substitution of a lysine near the active site resulted in 20% more activity with 53% less enzyme immobilized, thus increasing the specific activity of the decorated surface 2.5 fold. v DEDICATION This dissertation would not have been possible without the love and support of my family. vi ACKNOWLEDGEMENTS I am grateful for the help and support of everybody that contributed to my research. I would like to thank Dr. Wild, a teacher and friend, for giving me the freedom to pursue the things that I found most interesting, and to go outside the lab and work with friends and colleagues. It truly made my learning experience great. I owe a tremendous amount of my success to Dr. Janet Grimsley, for getting me started and keeping me on track. I would like to thank all the graduate students and undergraduates that worked with me. Dr. Shane Gold, Dr. Mauricio Rodriquez, Dr. Scott Pinkerton, David Armstrong, Rory Kern, Leamon Viveros, Jess Miller, Claire Rowe, Elizabeth Sherwood and Lisa Torres. Dr. Melinda Wales, her leadership in the lab was invaluable and her help was indispensable. Thank you. Finally I would like to thank Dr. Alex Simonian and Sheetal Paliwal for their help in accomplishing the sensor component of this dissertation. vii TABLE OF CONTENTS Page ABSTRACT ..................................................................................................................... iii DEDICATION ...................................................................................................................v ACKNOWLEDGEMENTS ..............................................................................................vi TABLE OF CONTENTS.................................................................................................vii LIST OF FIGURES...........................................................................................................ix LIST OF TABLES ............................................................................................................xi INTRODUCTION..............................................................................................................1 History of organophosphorus chemical warfare agents .................................................1 History of detection and decontamination options for CWA.........................................5 Enzymes: alternatives to caustic decontamination processes ........................................7 OP degrading enzymes...................................................................................................8 Metals and stability ......................................................................................................14 Rational design vs. directed evolution .........................................................................15 Rational design of enzymes for sensor surfaces...........................................................17 Research study objectives ............................................................................................19 MATERIALS AND METHODS .....................................................................................21 Variant construction and mutagenesis..........................................................................21 DNA sequencing ..........................................................................................................21 Plasmid mini-prep ........................................................................................................22 DH5α transformation ...................................................................................................23 Enzyme purification .....................................................................................................23 Metal chelation and reconstitution...............................................................................24 Equilibrium chemical denaturation..............................................................................24 Thermal denaturation ...................................................................................................25 Tryptophan excitation ..................................................................................................26 Thermolysin Digestion of OPH ...................................................................................26 Enzyme surface characterization..................................................................................27 Enzyme biotinylation ...................................................................................................27 OPH enzymatic activity assay......................................................................................28 Immobilized enzyme activity.......................................................................................29 viii Page Analysis of OPH activity with G agents ......................................................................29 SPREETA preparation and sensing layer construction................................................30 RESULTS.........................................................................................................................32 Wild type stability and metals......................................................................................32 Characterization of the native OPH enzyme and 254/257 variants .............................32 Design and characterization of a more stable P-S hydrolyzing enzyme ......................41 Probing the tertiary structure........................................................................................48 Kinetic comparison with nerve agents .........................................................................50 DISCUSSION ..................................................................................................................62 Catalysis and stability...................................................................................................62 Unfolding model ..........................................................................................................68 Designing enzymes for developing reactive surfaces ..................................................72 Protein design considerations.......................................................................................74 CONCLUSIONS..............................................................................................................77 REFERENCES.................................................................................................................79 VITA ................................................................................................................................87
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