THE MECHANISMS OF ARSENATE-ACTIVATION IN ENZYMATIC REACTIONS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By DONALD HILLMAN SLOCUM, B. S., M. S ****** The Ohio State University 1958 Approved by Adviser Department of Agricultural Biochemistry PREFACE Happy is the man that findeth wisdom, and the man that getteth understanding. For the merchandise of it is feetter than the merchandise of silver, and the gain thereof than fine gold. Proverbs, 3s 13 "• I1* ii ACKNOWLEDGMENTS The author wishes to express his gratitude and appreciation to Dr. Joseph E. Varner, whose guidance and patience were invaluable in the completion of this work, to Dr. George C. Webster for his counsel on phases of this problem, and to Mrs. June M. Slocum for her editorial critique during the preparation of this manuscript. iii TABLE OF CONTENTS Page IN TR ODUC TI ON 1 REVIEW OF LITERATURE *. ........ ............ 3 METHODS AND MATERIALS.............. 9 I . Enzyme Isolations 9 II. Reaction Requirements ............... 9 III. Isolations for 0xygen-l8 Measurements...,,, 11 IV. Synthesis of Intermediates. „ 13 ABBREVIATIONS...................... 16 EXPERIMENTAL RESULTS .......... 17 I. Homogeneity of Glutamine Synthetase. 17 II. Oxygen-18 Exchange in the Glutamine Synthetase Reaction........... 17 III. Oxygen-18 Exchange in the Arsenolysis of Glycogen by Muscle Phosphorylase..... 21 IV. Oxygen-18 Exchange in the Arsenate- Activated Hydrolysis of Citrulline by Ornithine Carbamyl Transferase 24 V. Oxygen-18 Exchange in the Arsenolysis of Acetyl Phosphate by Phosphoglycer- aldehyde Dehydrogenase ....... 24 VI. Oxygen-18 Exchange In the Fumarase Catalyzed Reaction Producing Malate 26 VII. Arsenolysis and Phosphorolysis of Potassium Cyanate......... 29 VIII. Identification of Acetyl Arsenate....... 36 IX. Effect of Arsenate and Phosphate Upon Urease Activity ............ 39 X. Examination of Urease Activity.......... 42 iv TABLE OP CONTENTS (continued) Page XI. Oxygen-18 Exchange during Decomposition of Urea in the Presence of Arsenate and Phosphate.......... 46 DISCUSSION..................... 49 SUMMARY......................................... 68 BIBLIOGRAPHY.............. 70 v LIST OP TABLES Page I. Glutamylhydroxamate Synthesis by Analytical Ultracentrifuge Preparations of Glutamine Synthetase.................... 20 II. Transfer of 0xygen-l8 during the Phosphorol- ysis and Arsenolysis of Glutamine............ 22 III. Transfer of 0xygen-l8 during the Arsenolytic Degradation of Glycogen .............. 23 IV. Transfer of Oxygen-18 in the Arsenolysis of Citrulline......... 23 V. Transfer of Oxygen-18 during the Arsenolysis of Acetyl Phosphate............ 27 VI. Transfer of Oxygen-18 during the Hydration of Fumarate............ 28 VII. Concentration Dependence in the Arsenolysis and Phosphorolysis of Potassium Cyanate...... 30 VIII. Decomposition of Carbamyl Phosphate in the Presence of Arsenate and Phosphate........... 33 IX. Effect of the Addition of High Concentration of Anion to Depleted Reaction Mixture 34 X. Arsenolytic Breakdown of Citrulline........... 35 XI. Identification of Acetyl Arsenate............ 37 XII. Determination of Carbamate Formation in Urea Breakdown by Urease ........ 40 XIII. Carbon Dioxide Production In the Urease Degradation of Urea........... 41 XIV. Hydrolysis of Carbamyl Phosphate by Urease 43 XV. Hydrolysis of Citrulline by Urease............ 44 XVI. Exchange of 0xygen-l8 from Arsenate and Phosphate during the Urease Decomposition of Urea ................. 47 vi LIST OP FIGURES Page 1. Photographs of the electrophoretic pattern of glutamine synthetase at pH 7.4 in Tris buffer with 0.1 ionic strength............. 18 2. Photographs of the ultracentrifuge pattern of glutamine synthetase in water at pH 7.1 with a protein concentration of 1.1 per cent........ 19 3. Dependence of phosphate and arsenate concen­ tration upon carbon dioxide production in the reaction of the anion with potassium cyanate ........................... 32 4. Infrared spectra of acetyl phosphate and acetyl arsenate...... 38 vii INTRODUCTION Compounds of arsenic have long been recognized as lethal. Among the various inorganic and organic mole­ cules which contain arsenic, the effect of the arsenate anion appears to be the most clearly defined. Because of its similarity in structure and reactivity to phosphate, a critical anion to normal life and function, its dele­ terious effects upon phosphate metabolism are not very surprising. Hypotheses have been presented to account for the effects upon enzymatic phosphorolysis• The generally ac­ cepted view is that arsenate replaces phosphate in the formation of essential phosphate esters. Because of the instability of the resulting arsenate homologs, the normal metabolic sequence of reactions is broken. The end product may often be inactive toward further metab­ olism in the non-esterified or unnatural form. An important criterion of this postulate is the formation of labile arsenate esters or anhydrides. Despite the fleeting formation of phosphate esters in some cases, the analogous arsenates may be similarly involved in the enzymatically catalyzed transformation. It is proposed in this work that the arsenate esters, 1 either free or enzyme bound., are formed during arsenoly­ tic degradation of many compounds and that these esters can he synthesized and characterizedc It has also been proposed that there is a universality of mechanism in the arsenate-activated hydrolyses. Among the enzyme-substrate systems studied are glutamine synthetase-glutamine, muscle phosphorylase- glycogen, ornithine carbamyl transferase-citrulline, and phosphoglyceraldehyde dehydrogenase-acetyl phosphate. The compounds synthesized during this study are carbamyl arsenate, mono- and tri-acetyl and mono- and tri­ benzoyl arsenates. Allied experiments carried out -with special emphasis on their relation to the arsenolytic reactions mentioned above include the arsenolysis of potassium cyanate and the effect of arsenate upon the action of urease and fumarase. 2 REVIEW OP THE LITERATURE In 1932 Harden wrote, "The close analogy which exists between the chemical functions of phosphorus and arsenic lends some interest to the examination of the effect of arsenate upon yeast juice" (1). It had been shown by Harden and Young (2, 3) that arsenate produced considerable acceleration of the fermentation process in yeast extract. This alteration in rate was maintained for a considerable period and was independent of arsenate concentration. They also reported that no organic arsenate esters corresponding to the hexosephosphates appeared to be formed. It has been suggested that arsenate esters are formed in small quantity and are rapidly hydrolized; therefore isolation is not possible. At that time, Harden mentioned that though total fermentation with arsenate exceeds that with phosphate, fer­ mentation requires phosphate and cannot proceed in the presence of arsenate in the total absence of phosphate. Since Harden and Young (2) discovered the amazingly enormous increase in the fermentation of hexose-diphosphate by arsenate, the subject has frequently attracted investi­ gators to examine the steps of the fermentation chain for possible site of activity of arsenate. Braunstein, in a series of papers from 1931 to 193^ (*J-> 5> 6, 7* 8, 9), studied the effect of arsenate on glycolyzing erythrocytes. Among the interesting findings were the evidence for bound 3 arsenate in an acid labile form, the parallelism of vana­ date to arsenate in stimulatory action and the preparation of a fructose arsenate compound of possible polymeric character* He was in accord with the proposals of Harden (1) regarding the possible existence of the labile hexose- arsenate intermediates. The first advance concerning the specific effects of arsenate was made by Meyerhof (10) and Meyerhof and Kiessling (11) who found that the primary action in glycolysis and fermentation occurs during the transformation of triose phosphate to phosphoglyceric acid. It was shown later that phosphoglyceraldehyde is oxidized in the presence of phosphate with subsequent production of ATP (12, 13, 1^). However, when arsenate is substituted for phosphate, the oxidation proceeds with equal speed while in the presence of both anions, the rate of oxidation is unchanged and the phosphate uptake is reduced to zero. Warburg and Christian (15, 16) and Negelein and Brorael (17, 1 8) showed that the reversible stoiciometric coupling reaction forming ATP could be traced to a 1, 3-diphospho- glyeerie acid intermediate. Warburg and Christian (16) proposed that the observed effect of arsenate was due to the spontaneous decomposition of the 1-arseno compound. They theorized that the hydrolysis proceeds because of the ar­ senate instability in contrast to the relative stability of the phosphate counterparts. This explanation, though lack­ ing any direct experimental evidence, may be accepted as very probable (19). Doudoroff et al. (20) extended this concept when they found that glucose-l-phosphate was de­ graded in the presence of arsenate and the enzyme, sucrose phosphorylase. The product is glucose and no arsenate ester accumulates. Sucrose underwent the same “arsenolytic decomposition", a term introduced by these researchers. Similar work was done with potato phosphorylase (21) and with muscle phosphorylase (22). Following these reports was the investigation of the arsenate-activated decompo­ sition of acetyl phosphate (23,