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Study of Nucleoside Degrading Enzyme Activities in Bean, Organic Bean, Okra, Organic Okra, Squash and Organic Squash

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Study of Nucleoside Degrading Enzyme Activities in Bean, Organic Bean, Okra, Organic Okra, Squash and Organic Squash STUDY OF NUCLEOSIDE DEGRADING ENZYME ACTIVITIES IN BEAN, ORGANIC BEAN, OKRA, ORGANIC OKRA, SQUASH AND ORGANIC SQUASH by Shafiqa A. Alshaiban A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in Chemistry Middle Tennessee State University August 2016 Thesis Committee: Dr. Paul C. Kline, Chair Dr. Andrew Burden Dr. Anthony Farone I dedicate this research to my parents, my sisters, and my brothers. I love you all. ii ACKNOWLEDGEMENTS I would like to sincerely thank Dr. Paul Kline for the support and guidance he has offered throughout the entirety of the research and thesis writing process. I also wish to thank my committee members, Dr. Donald A. Burden and Dr. Anthony Farone for their advice and willingness to read my work. I would like to thank all the staff and faculty members for their valuable support. Finally, I would like to express my thanks to my family and my friends for their support and love. Special thanks to my loving parents, my brothers and my sisters for their prayer, concern and kind words over the years. iii ABSTRACT Pyrimidine and purine nucleotide metabolism are essential for development and growth of all organisms. Nucleoside degradation reactions have been found in virtually all organisms. Many enzymes are involved in the degradation and salvage of nucleotides, nucleobases and nucleosides. Deaminases contribute in interconversion of one nucleoside into another by removing amino groups from the base. Nucleoside hydrolase is a glycosidase that catalyzes the cleavage of the N-glycosidic bond in nucleosides to facilitate recycling of nucleobases. The nucleoside degrading enzyme activity was tested by adding a crude protein extract from 5-9 day old bean, organic bean, okra, organic okra, squash and organic squash seedlings to reaction mixtures containing either inosine, uridine, cytidine, adenosine, or 2'-deoxyadenosine. The activities of the enzyme were determined on a Dionex Ultimate 3000 High Performance Liquid chromatography system (HPLC). Nucleosidases were found to be active in all six plants and specific activities are reported. There are variations in the level of activities observed between bean, organic bean, okra, organic okra, squash and organic squash. Based on the specific activities the highest activity was recorded for uridine, cytidine and adenosine in different types of seeds while inosine and 2'deoxyadenosine had the lowest activity. Bean was the lowest in activity with all five nucleosidases compared to nucleosidase metabolizing activities in organic bean, okra, organic okra, squash and organic squash. iv TABLE OF CONTENTS PAGE LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii CHAPTER I: INTRODUCTION……………..............................................................................1 Purines and Pyrimidines.................................................................................................................. 2 Purine and Pyrimidine Metabolism ................................................................................................ 4 Nucleoside Hydrolases.................................................................................................................. 11 Uridine Nucleosidase..................................................................................................................... 17 Inosine Nucleosidase…................................................................................................................. 17 Adenosine Nucleosidase ............................................................................................................... 18 Cytidine Nucleosidase .................................................................................................................. 20 Nucleoside Deaminases ................................................................................................................ 20 Current Research ........................................................................................................................... 22 CHAPTER II: MATERIALS AND METHODS ........................................................................ 23 Equipment and Instrumentation .................................................................................................... 23 Materials and Reagents.................................................................................................................. 23 Methods......................................................................................................................................... 23 Measurement of Enzyme Activity. ............................................................................................... 24 Determination of Protein Concentration ....................................................................................... 25 CHAPTER III: RESULTS AND DISCUSSION…………………...………………..…………26 Enzyme Activities in Okra and Organic Okra. ............................................................................. 30 Enzyme Activities in Bean and Organic Bean ..............................................................................44 Enzyme Activities in Squash and Organic squash…………………….…………………………58 CHAPTER IV: CONCLUSIONS………………………………………………………..……...71 REFERENCES ..............................................................................................................................73 v LIST OF TABLES TABLE PAGE 1. Summary of retention times of nucleosides and nucleobase…………….…………................29 2. Nucleosidase and deaminase activities in okra………………………………….………….....43 3. Nucleosidase and deaminase activities in organic okra…………………………….……........43 4. Nucleosidase and deaminase activities in bean……………………………………………… 57 5. Nucleosidase and deaminase activities in organic bean………………………….…………...57 6. Nucleosidase and deaminase activities in squash………………………………………......…70 7. Nucleosidase and deaminase activities in organic squash…………...………………………..70 vi LIST OF FIGURES FIGURE PAGE 1. Structural elements of nucleosides and nucleotides…………………………….......................1 2. The structures of ribose in RNA and 2-deoxyribose in DNA………...……………………….2 3. Common purine and pyrimidine bases with their numbering schemes……………...……..….3 4. A general diagram of plant pyrimidine metabolism……………………………………...........5 5. A general diagram of plant purine metabolism………………...……………………………...8 6. Chemical structures of the mentioned unusual bases…...……………………........................11 7. Different quaternary nucleoside hydrolase structures are built from a common fold…....…..15 8. Comparison between the active sites...……...…………………………………………....…..16 9. Hydrolysis of uridine to uracil and ribose by uridine nucleosidase………………………......17 10. Hydrolysis of inosine to hypoxanthine and ribose by inosine nucleosidase………………....18 11. Hydrolysis of adenosine to adenine and ribose by adenosine nucleosidase.............................19 12. Hydrolysis of cytidine to cytosine and ribose by cytidine nucleosidase..................................20 13. Adenosine deaminase converts adenosine to inosine and releases NH 3.............. ………........21 14. Cytidine deaminase converts cytidine to uridine and releases NH 3.........................................22 15. Protein concentrations calibration curve..................................................................................25 16. Hydrolysis of deoxyadenosine to adenine and deoxyribose by deoxyadenosine nucleosidase…………………………………………………………..……………………....27 17. Structures of additional nucleosides and bases........................................................................28 18. Uridine to uracil conversion catalyzed by uridine nucleosidase in okra..................................32 19. Uridine to uracil conversion catalyzed by uridine nucleosidase in organic okra.....................33 20. Inosine to hypoxanthine conversion catalyzed by inosine nucleosidase in okra.....................34 21. Inosine to hypoxanthine conversion catalyzed by inosine nucleosidase in organic okra........35 22. Cytidine degradation reaction in okra......................................................................................36 23. Cytidine degradation reaction in organic okra.........................................................................37 24. Adenosine breakdown reaction in okra...................................................................................39 25. Adenosine breakdown reaction in organic okra......................................................................40 vii 26. 2'-Deoxyadenosine degrading reaction in okra.......................................................................41 27. 2'-Deoxyadenosine degradation reaction in organic okra ......................................................42 28. Uridine to uracil conversion catalyzed by uridine nucleosidase in bean ...............................45 29. Uridine to uracil conversion catalyzed by uridine nucleosidase in organic bean ..................46 30. Adenosine degradation reaction in bean.................................................................................48 31. Adenosine degradation reaction in organic bean....................................................................49 32. 2'-Deoxyadenosine breakdown reaction in bean.....................................................................50
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