INFORMATION TO USERS The most advanced technology has been used to photo­ graph and reproduce this manuscript from the microfilm master. UMI films the original text directly from the copy submitted. Thus, some dissertation copies are in typewriter face, while others may be from a computer printer. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyrighted material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are re­ produced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each oversize page is available as one exposure on a standard 35 mm slide or as a 17" x 23" black and white photographic print for an additional charge. Photographs included in the original manuscript have been reproduced xerographically in this copy. 35 mm slides or 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Accessing the World'sUMI Information since 1938 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA Order Number 8820378 Stereochemical studies in anaerobic metabolism Zydowsky, Lynne Douthit, Ph.D. The Ohio State University, 1988 UMI 300 N. Zeeb Rd. Ann Aibor, M I 48106 PLEASE NOTE: In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V . 1. Glossy photographs or pages_____ 2. Colored illustrations, paper or print_______ 3. Photographs with dark background_____ 4. Illustrations are poor copy_______ 5. Pages with black marks, not original copy ______ 6. Print shows through as there is text on both sides of page _______ 7. Indistinct, broken or small print on several pages. 8. Print exceeds margin requirements ______ 9. Tightly bound copy with print lost in spine _______ 10. Computer printout pages with indistinct print _______ 11. Page(s)___________ lacking when material received, and not available from school or author. 12. Page(s) seem to be missing in numbering only as text follows. 13. Two pages numbered . Text follows. 14. Curling and wrinkled pages 15. Dissertation contains pages with print at a slant, filmed as received 16. Other UMI STEREOCHEMICAL STUDIES IN ANAEROBIC METABOLISM DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Lynne Douthit Zydowsky, B.S. The Ohio State University 1988 Dissertation Committee: Approved By Heinz G. Floss Michael H. Klapper Robert M. Mayer Adviser Ming-Daw Tsai Department of Chemistry To Tom, Who is both my husband and best friend ii ACKNOWLEDGMENTS I would like to express my sincere appreciation to my research adviser, Dr. Heinz G. Floss, for his support and guidance during my stay in his research group. He gave me the opportunity to join his group even though he knew that my chemical backgound and research skills needed substantial development. I will always be greatly indebted to him for taking this chance with me. I would like thank Dr. John N Reeve for opening his laboratory to me and providing research assistance along the way. I would also like to thank my dissertation committee members; Dr. Michael H. Klapper, Dr. Robert M. Mayer, and Dr. Ming-Daw Tsai for their patience during the final stages of my graduate career. I am indebted to my coworkers in the Floss group. They offered me the scientific assistance and friendship which made my stay at The Ohio State University a rewarding and enjoyable experience. A special thanks goes to Jim Brown and Elizabeth Haas. They offered me not only their sceintific knowledge, but also their home and their hearts at a time when I needed both. Dr. Leroy Salerni, my friend and former professor from Butler University, did not let me settle for second best. He started my graduate career in the right direction by suggesting that I go to Purdue University and work for Dr. Heinz G. Floss. I will always be thankful for his advice and confidence in me. To Tom Zydowsky, my husband and best friend, I owe a very special thanks. His selfless and loving support, both scientific and personal, was invaluable to me. He offered suggestions and comments which helped me scientifically, and gave me the support and encouragement to continue in pursuit of my career. For this I will always love him. iv VITA May 22,1958 ............................................................ Born - Olney, Illinois May, 1 9 8 1................................................................. B.S., Butler University, Indianapolis, Indiana August, 1981 - December, 1982 ...................... Graduate Student PurdueUniversity, West Lafayette, Indiana January, 1983 - January 1987 Graduate Research November 1987 - present Associate in the Department of Chemistry, The Ohio State University Feburary 1987 - November 1987 ....................... Proctor and Gamble Predoctoral Fellowship PUBLICATIONS 1. L.D. Zydowsky, T.M. Zydowsky, E.S. Haas, J.W. Brown, J.N. Reeve and H.G. Floss. "Stereochemical Course of Methyl Transfer from Methanol to Methyl Coenzyme M in Cell-Free Extracts of Methanosarcina barkeri." J. Am. Chem. Soc,. 1987. 109. 7922. 2. S. Raybuck, N.R. Bastian, L.D. Zydowsky, K.Kobayashi, H.G. Floss, W.H. Orme-Johnson and C.T. Walsh. "Nickel-Containing CO Dehydrogenase Catalyses Reversible Decarbonylation of Acetyl CoA with Retention of Stereochemistry at the Methyl Group." J. Am. Chem. Soc. 1987. 109. 3171. v 3. H. Lebertz, H. Simon, L.F. Courtney, S.J. Benkovic, L.D. Zydowsky, K. Lee and H.G. Floss. "Stereochemistry of Acetic Acid Formation from 5-Methyl Tetrahydrofolate by Clostridium thermoaceticum." J. Am. Chem. Soc. 1987. 109. 3173. FIELDS OF STUDY Bioorganic Chemistry and Mechanistic Enzymology. TABLE OF CONTENTS PAGE DEDICATION i i ACKNOWLEDGMENTS.......................................................................................... i i i VITA............................................................................................................................. v LIST OF FIGURES.................................................................................................... x i LIST OF ABBREVIATIONS................................................................................... x iv PART I INTRODUCTION..............................................................................................1 RESULTS AND DISCUSSION............................................................................... 10 Preparation of (R)- and (S)-[ 2H i, 3H]Acetyl Coenzyme A ............................................................................................. 1 0 The Carbonylation Reaction with Purified Carbon Monoxide Dehydrogenase (CODH) from Clostridium thermoaceticum ......................................................................................... 11 Stereochemical Analysis of Acetic Acid Formation from (Methyl-R)- and (Methyl-S)-[ 2 H i, 3 H]methyltetra- hydrofolate by a Cell-Free Extract of Clostridium thermoaceticum. 1 3 CONCLUSION........................................................................................................ 16 EXPERIMENTAL..................................................................................................... 20 Materials and Methods ............................................................................ 2 0 Synthesis of (R)- and (S)-[ 2H-| 3H]Acetyl Coenzyme A 21 Carbonylation Exchange with Purified CO Dehydro­ genase (CODH) from Clostridium thermoaceticum with (R)- and (S)-[ 2H-| 3H]Acetyl Coenzyme A ................................ 22 Conversion of (Methyl-R)- and (Methyl-S)- [2H i ,3 H]methyltetrahydrofolate with the Cell-Free Extract From Clostridium thermoaceticum ...................................... 23 PART II INTRODUCTION...................................................................................... 24 Methanogenesis from CO 2 and H 2.................................................... 27 Methanogenesis from Methanol, Methylamine and Acetate ....................................................................................................... 31 RESULTS AND DISCUSSION................................................................................ 3 6 Cultivation of Methanosarcina barkeri on Methanol Methylamine, and Acetate ....................................................................... 3 6 Preparation of an Active Cell-Free Extract from Methanosarcina barkeri ............................................................................ 3 9 Conversion of CD 3OH to CD3H with the Cell-Free Extract of Methanosarcina barkeri ........................................................ 39 Synthesis of (R)- and (SJ-pH-j , 3H]Methanol ..................................... 40 Conversion of (R)- and (S)-[ 2H-| ,3H]Methanol (lk) and (la) to Acetate for Configurational Analysis ........................ 52 Conversion of 14C-Methanol into Methyl Coenzyme M using the Cell-Free Extract of Methanosarcina barkeri and Isolation of Methyl Coenzyme M .................................................... 5 9 Conversion of (R)- and (S)-[ 2H i, 3 H]Methanol into (R)- and (S)-[2H i, 3H]Methyl Coenzyme M with the Cell-Free Extract of Methanosarcina barkeri ....................................................... 60 Degradation of [ 2H-|,3H]Methyl