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Cooperative Interactions in Glutamic Acid Decarboxylase David Lavern Witte Iowa State University

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Cooperative Interactions in Glutamic Acid Decarboxylase David Lavern Witte Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1971 Cooperative interactions in glutamic acid decarboxylase David Lavern Witte Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biochemistry Commons Recommended Citation Witte, David Lavern, "Cooperative interactions in glutamic acid decarboxylase " (1971). Retrospective Theses and Dissertations. 4930. https://lib.dr.iastate.edu/rtd/4930 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. 71-26,904 WITTE, David Lavem, 1943- COOPERATIVE INTERACTIONS IN GLUTAMIC ACID DECARBOXYLASE. Iowa State University, Ph.D., 1971 Biochemistiy University Microfilms, A XEROX Company, Ann Arbor, Michigan THIS DISSERTATION HAS BEQf MICROFILMED EXACTLY AS RECEIVED Cooperative interactions in glutamic acid decarboxylase by David Lavern Witte A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of The Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Subject: Biochemistry Approved: Signature was redacted for privacy. Signature was redacted for privacy. H tment Signature was redacted for privacy. f Graduée College Iowa State University Of Science and Technology Ames, Iowa 1971 ii TABLE OF CONTENTS Page DEDICATION i i i ABBREVIATIONS iv INTRODUCTION 1 LITERATURE REVIEW 2 EXPERIMENTAL 15 RESULTS AND DISCUSSION 27 LITERATURE CITED 75 ACKNOWLEDGMENTS 78 APPENDIX 79 DEDICATION To iny wife iv ABBREVIATIONS ATCC - American Type Culture Collection CD - circular dichroistn DEAE - diethyl amino ethyl DTT - dithio threitol (CIel and's reagent) E. coVi - Esdherichla coVt GABA - gama amino butyric acid GAD - glutamic acid decarboxylase GOT - glutamate oxalacetate transaminase PLP - pyridoxal 5' phosphate 1 INTRODUCTION Glutamic acid is a metabolite of central importance. This work will be concerned with only one of glutamate's various metabolic fates, alpha decarboxylation, which is catalyzed by glutamic acid decarboxylase (GAD). GAD is present in bacterial and mammalian systems and the literature on these systems will be reviewed. However, this work is concerned with GAD from E. coH ATCC 11246. Improved methods of bacterial culture and GAD isolation will be presented. The velocity of GAD is regulated by anionic species and the mechanism of this activation will be considered. It is also known that GAD is a hexameric protein of identical subunits arranged in an octahedral array. An oligomeric structure of this type often leads to a complex kinetic pattern. The accepted ideas of enzymic regulation through the interaction of protein subunits will be reviewed and then extended to include the inherent asymmetry of the subunits. The ultimate goal is to correlate the observed kinetic properties of GAD with the known structural properties. 2 LITERATURE REVIEW The presence of amino acid decarboxylases was first suggested to explain the presence of the large number of amines found in the cultures of putrefying bacteria (Ackermann, 1911). If these.bacteria were cultured in amino acid rich media, the amino acids were converted to the corresponding amines, i.e., tyramine, histamine, etc. Later work (Gale, 1941) showed these amines were produced by the action of six amino acid decarboxylases. Gale and his coworkers engaged in an extensive investigation of the bacterial decarboxylases (Gale, 1946). They found that the enzymes all had acidic pH optima and they were quite specific for their amino acid substrates. Gale also elucidated the physiological conditions necessary for the production of the decarboxylases. Gale stated that to produce a specific decarboxylase (1) the bacteria must be cultured on the substrate for that enzyme, (2) the enzyme will not be synthesized during the initial stages of the culture, (3) the medium must become acidic and (4) the decarboxylase activity will be fully developed only after cessation of log phase growth. After the initial work of Gale, most of the subsequent work was devoted to the partial purification of the individual decarboxylases. The enzymes were purified only to the extent of removal of other decarboxylases. These crude preparations were then used to analyze amino acid mixtures and selectively destroy L-amino acids in a racemic mixture. Substrate specific­ ities, optimal conditions and kinetic parameters were also determined with these crude preparations. This early work .was reviewed by Gale (1946) and provided a sound foundation for the investigators to follow. 3 About this time considerable interest was focused on the cofactor of the decarboxylases. Gunsalus and co-workers (1944) identified the active cofactor of tyrosine decarboxylase as PLP, results which were confirmed by Taylor and Gale (1945). This finding initiated a renewed interest and investigation into the nature of the mechanism of the decarboxylases. Ultimately, two mechanisms were proposed, both involving the formation of a Schiff's base between PLP and the amino acid substrate. The Werle and Koch (1949) mechanism involved the labilization of the alpha proton and predicted the incorporation of two solvent hydrogens into the product amine. The second mechanism, proposed independently by Metzler et al. (1954), Braunstein and Shemyakin (1953) and by Mandeles et al. (1954), did not involve labilization of the alpha hydrogen and thus predicted the incor­ poration of only one solvent hydrogen into the product amine. The later proposal was confirmed using DgO by Mandeles et al. (1954). Several recent findings should be mentioned. First, Dunathan (1966) proposed that the alpha bond which is to be labilized in the amino acid substrate must be. perpendicular to the aromatic system of the pyridoxal imine. This stereochemical arrangement ccuses a gain in delocalization energy which aids in the bond breaking process. Secondly, the results of Huntley and Metzler (1968) with the reaction of alpha methyl glutamate with GAD showed that this substrate analog could stereo chemically distort the active site sufficiently to cause an error in proton transfer. This error allowed the decarboxylation dependent transamination of glutamate. However, the rate of this transamination reaction was 100 times less than that of the normal decarboxylation of alpha methyl glutamate. Finally, an exception should be noted. Snell and Riley (1970) have shown that histidine 4 decarboxylase does not contain PLP. Instead, the active carbonyl group is provided by an N-terminal pyruvate. The physiological function and the control of the synthesis of the decarboxylases remains an open question. The early work had given rise to two hypotheses. Neither has been confirmed. Noting that decarboxylases were formed only under acidic culture conditions, Koessler and Hanke (1919) proposed that decarboxylases provided a protection system. The decarboxy­ lases produced basic amines and CO2 which would help buffer the medium at a higher pH and thus protect the cells from the hardships of an acidic medium. Alternately, Gale (1946) proposed that the decarboxylases served to increase the CO2 concentration available to the cells so that the cells could carry out those processes which required CO2. This was necessary since CO2 is evolved from an acidic medium. More recently, a research group headed by Hal pern has undertaken the investigation of the use of glutamate by E. ooH. Halpern (1962) has shown that GAD activity is sensitive to repression by succinate and pyruvate but does not show glucose repression, which is somewhat unusual. In order to understand this observation and the earlier observations of substrate induction and the effects of pH and culture age on decarboxylase production, Halpern et ai. undertook an extensive genetic investigation (Halpern et aZ., 1965, 1967, 1969, 1970). It was found that glutamate permease involved three genes, gltS, the structural gene, gltR, the regulator gene, and gltC, the operator. Several mutants were isolated that were unable to decarboxy- late glutamate due to a faulty permease system. Also, it was found that some mutants could not grow on glutamate as a sole carbon source if they contained high levels of GAD, because they lacked the enzymes to use the 5 product of GAD, GABA. It is also interesting that the kinetics of glutamate permease showed a non-linearity with respect to glutamate similar to that reported for GAD in this work (i.e., an activation at high glutamate levels). Halpern (1970) also reported that GAD is not under the control of gltR and the GAD system involves two genes, gadR, a regulator, and gadS, the structural gene. The wild type allele, gadR^, shows partial expression of gadS and the mutant, gadR", shows full expression of gadS. However, the nature of the gadR and gltR gene products is still unknown and therefore the details of the control of glutamate metabolism and the physiological significance of these enzymes are still under investigation. The interest in decarboxylases seemed to wane in the late 1950's. Perhaps because decarboxylases did not readily lend themselves to the more precise and convenient spectrophotometric assay methods, but were only suited to the classical manometric methods. However, Shukuya and Schwert (1960) reported a large body of significant
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