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Characterization of a Decarboxylation - Dependent Transaminase and the Metabolism of Alpha- Aminoisobutyric Acid

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Characterization of a Decarboxylation - Dependent Transaminase and the Metabolism of Alpha- Aminoisobutyric Acid Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1971 Characterization of a Decarboxylation - Dependent Transaminase and the Metabolism of Alpha- Aminoisobutyric Acid. Gaston Orlando Daumy Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Daumy, Gaston Orlando, "Characterization of a Decarboxylation - Dependent Transaminase and the Metabolism of Alpha- Aminoisobutyric Acid." (1971). LSU Historical Dissertations and Theses. 1915. https://digitalcommons.lsu.edu/gradschool_disstheses/1915 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. DAUMY, Gaston Orlando, 1944- CHARACTERIZATION .OF A DECARBOXYLATION-DEPENDENT TRANSAMINASE AND THE METABOLISM OF ALPHA- AMINOISOBUTYRIC ACID. The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1971 Microbiology University Microfilms, A XEROX Company, Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED CHARACTERIZATION OF A DECARBOXYLATION-DEPENDENT TRANSAMINASE AND THE METABOLISM OF ALPHA- AMINOISOBUTYRIC ACID A dissertation Submitted to the Graduate Faculty o f the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements fcr che degree of Doctor of Philosophy in The Department of Microbiology by Gaston Orlando Daumy B.S., Louisiana State University, 1967 January, 1971 ACKNOWLEDGMENT The author wishes to express his appreciation to the faculty members of the Departments of Microbiology and Biochemistry as well as his fellow students, who in their own way aided in this research. He is especially grateful to his mentor, Dr. A. D. Larson, for his excellent guidance, encouragement and criticism throughout this investigation. He also wishes to thank Dr. H. D. Braymer and Dr. F. W. Forney for the endless discussions and arguments they held with the author which resulted in valuable contribu­ tions to this dissertation. The author is very appreciative for the predoc- toral traineeship awarded through the Department of ‘ • Public Health Services training Grant No. 5TI-GM00692 from the National Institute of General Medical Science. Finally, the author wishes to thank his family for their reassurance and encouragement throughout his tenure in graduate school. This dissertation is dedicated to his wife, Mimr, for the aid and comfort she provided the author during his graduate studies and for undertaking and fulfilling the tremendous task of typing this dissertation. TABLE OF CONTENTS Page ACKNOWLEDGMENT ............................... - ii TABLE OF CONTENTS ............................... iii LIST OF ABBREVIATIONS ...... V LIST OF TABLES .................................. vii LIST OF FIGURES ................................. ix ABSTRACT ........................ xi INTRODUCTION .................................... 1 REVIEW OF LITERATURE ............................ 3 MATERIALS AND METHODS .......................... 15 Chemicals .......... 15 Organism .................................... 16 Culturing the Organism ......... 17 Preparation of Cellular Extracts ....... IS Manometric Methods ......................... 19 Assays ...................................... 20 Enzyme Purification . ..................... 22 Enzyme Resolution ........................... 23 Analytical Gel Electrophoresis ............. 24 RESULTS ......................................... 25 Growth of the organism ..................... 25 Induction ................................... 36 Purification of Decarboxylation-Dependent Transaminase .............................. 39 Characterization of the Decarboxylation- Dependent Transaminase ................... 42 Enzyme resolution .................... 42 Mechanism of enzyme action ........... 4& Enzyme kinetics ...................... 60 Catabolism of AIB ........................... 70 DISCUSSION ...................................... 73 Page LITERATURE CITED t 82 VITA 91 LIST OF ABBREVIATIONS Ac acetate AIB oC-amino isobutyric acid C centigrade cm centimeter Enz enzyme: decarboxylation-dependent transam inase g gram x g gravity, centrifugal ■ v initial velocity ISPA isopropylamine 1 liter MeOH methanol MeAc methyl acetate Km Michaelis constant - - p.g microgram ;al microliter umoles micromoles mg milligram ml milliliter mM millimolar min minute M molar nm nanometer . OD optical density % percent PL pyridoxal PLP pyridoxal~5T-phosphate PM pyridoxamine PMP pyridoxamine-51-phosphate Py pyruvat e Py-ald pyruvic aldehyde rpm revolutions per minute S substrate concentration TCA trichloroacetic acid v/v volume per volume w/v weight per volume vi LIST OF TABLES TABLE Page 1. Growth of the organism with acetone as sole carbon source................... 32 2. Growth of the organism in nutrient broth plus acetone........... 33 3* Effect of combining acetone and AIB as carbon sources on decarboxyla­ tion-dependent transaminase produc­ tion. ............................ 37 4. Formation of decarboxylation-depen­ dent transaminase with AIB and/or NH^NO^ as nitrogen source . 3& 5. Formation of decarboxylation-depen­ dent transaminase in resting cells. 40 6. Specific activities of fractions during purification of the decar­ boxylation-dependent transaminase-.. 43 7. Resolution of the decarboxylation- dependent transaminase... 45 S. Effect of prolonged dialysis on decarboxylation-dependent transam­ inase............................... 46 9. Effect of pyruvate and AIB on the resolution of decarboxylation-depen­ dent transaminase.................. 47 10. Inability of PLP to replace pyru­ vate in the decarboxylation-depen­ dent transaminase.................. 49 11. Inability of PLP to replace pyru­ vate in the decarboxylation-depen­ dent transaminase in the-presence of catalytic amounts of the keto acid................................ 50 vii Q TABLE Page 12. Activity of decarboxylation-depen­ dent transaminase in the presence of varied amounts of acetone....... 55 13. Activity of decarboxylation-depen­ dent transaminase in the presence of varied amounts of ISPA.......... 57 14. Inability of the decarboxylation- dependent transaminase to transam- inate between ISPA and pyruvate.... 58 15. Ability of PMP to replace PLP in the decarboxylation-dependent transaminase system.......-......... 59 16. Oxidation of proposed intermediates in the catabolism of AIB........... 71 viii LIST OF FIGURES FIGURE Page 1. Growth curves of the organism in AIB minimal medium...................... 27 2. Effect of substrate (AIB) concentra­ tion (0.001 to 0.05$) on the growth of the organism..................... 29 3. Effect of substrate (AIB) concentra­ tion (0.01 to 0.5$) on the growth of the organism........................ 30 4. Growth curve of the organism in AIB minimal medium...................... 34 5. Sucrose density gradient centrifuga­ tion of apoensyme in the presence and absence of PLP.................. 52 6. Sodium borohydride reduction of holo- and apoenzyme................. 53 7. Qualitative assay for transamination using thin layer chromatography 61 ' Inability of the enzyme to transami- nate between PMP and pyruvate...... 62 9. Effect of pyruvate on the kinetics of the decarboxylation-dependent transaminase........................ 64 10. Kinetic plot of v vs S at constant pyruvate concentration (3-33 jimoles/ ml).................................. 65 11. Double reciprocal plot at constant pyruvate concentration (3.33 pmoles/ m l ).................................. 66 12. Kinetic plot of v vs S a t .constant AIB concentrations.................. 67 ix FIGURE Page 13. a) Kinetic plot v vs S and b) double reciprocal plot at constant AIB con­ centration (S.B2 jomoles/ml}........ 6$ 14. a) Kinetic plot v vs S and b) double reciprocal plot at constant AIB con­ centration (17.6 juunoles/ml}........ 69 x ABSTRACT The growth of a soil pseudomonad in a medium con­ taining c<-amino isobutyric acid as sole carbon source was investigated. Growth appeared to be limited either by failure of the medium to satisfy nutritional re­ quirements or by the production of some toxic products by the organism during growth. The precise cause or causes remained undetermined. The decarboxylation-dependent transaminase in­ volved in the degradation of oC-amino isobutyric acid was partially purified and the reaction mechanism characterised. The ensyme was purified 65-fold by combining protamine sulfate treatment, heat treatment, ammonium sulfate and acetone fractionation,•and Sepha-- dex gel filtration. The decarboxylation-dependent transaminase re­ quired stoichiometric amounts of pyruvate and catalytic amounts of pyridoxal-5 f-phosphate for activity. The resolved ensyme was reactivated with either pyridox- amine-51-phosphate or pyridoxal-5T-phosphate. The coenzyme was easily resolved from the holoenzyme by dialysis in the presence of the substrate. That pyridoxal-5T-phosphate participated in the enzymatic reaction as opposed to being a structural component of the enzyme was determined by sucrose density gra­ dient centrifugation of holo- and apoenzyme* Both enzyme forms sedimented at equal rates. Results ob­ tained with NaBH^ reduction of holo- and apoenzyme again indicated an active catalytic role for pyridoxal- 5’-phosphate.
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