103 927 THS '» ”mafia 1:" LIBRAIL y , lfiFm; ‘ .. Michigan State » University This is to certify that the thesis entitled The Role of H+ in the K+ Activation of Rabbit Muscle 5'AMP Aminohydrolase presented by John C.W. Campbell, Jr. has been accepted towards fulfillment of the requirements for M' S ' degree in Biochemistry Major‘professor X3 77 THE ROLE OF H+ IN THE K+ ACTIVATION 0F RABBIT MUSCLE 5'AMP AMINOHYDROLASE By John C. N. Campbell, Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Biochemistry 1977 ABSTRACT THE ROLE OF H+ IN THE K+ ACTIVATION 0F RABBIT MUSCLE 5'AMP AMINOHYDROLASE By John C. N. Campbell, Jr. This study is an examination of the role of H+ in the K+ mediated activation of 5'AMP aminohydrolase. Both kinetic and equilibrium experiments were performed in defining this role. Kinetic experiments consisted of observing changes in Km, V , and max the Hill slope as functions of pH, while equilibrium experiments observed changes in numbers of H+ bound to enzyme upon K+ binding. Data are presented which indicates that K+ binding is linked to H+ binding. Thus activation of enzyme is observed to take place when these H+ binding sites undergo either a change in degree of ioniza- tion or protonation. To My Parents ii ACKNOWLEDGMENTS I wish to acknowledge the assistance of Dr. Norman Good of the Botany Department. Also, the help and advice of Mark Brody, Ann Aust and Shyn-Long Yun. In addition, I especially wish to thank Dr. Clarence Suelter for his guidance and inspiration, without which this thesis would not have been possible. TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . vii LIST OF ABBREVIATIONS . . . . . . . . . . . . . . viii —I INTRODUCTION . LITERATURE REVIEW . . . . . Occurrence of 5'AMP aminohydrolase . Purification . . . . . . . . . Structure . Characterization As a Metalloenzyme Activation and Inhibition . Chemical Modifications Physiological Role . METHODS AND MATERIALS . Enzyme Purification Enzyme Assays . . Removal of Activating Cations . . . . . . . Protein Determination . . . . . . . . . l0 Proton Release and Uptake Experiments . . . . . . ll KA Determination . . . . . . . . . . . . . ll Reagents . . . . . . . . . . . . . . . . 12 Comm (I) (nap-wooden) N RESULTS . . . . . . . . . . . . . . . . . . l3 'Kinetic Constants for K+ ActivatiOn of 5'AMP aminohydrolase As a Function of pH . . . . . . l3 Release or Uptake of H+ Upon K+ Binding to 5' AMP aminohydrolase . . . . . . . l3 Stoichiometry of H+ Released or Absorbed Upon K+ Binding to 5' AMP aminohydrolase As a Function of pH . . . . . . . . . . . 23 DISCUSSION . . . . . . . . . . . . . . . . . 26 iv Page SUMMARY . . . . . . . . . . . — . . . . . . 43 Appendices . . . . . . . . . . . . . . . . . 44 A. DERIVATION OF EQUATION 2 OF THE DISCUSSION . . . 45 B. DERIVATION OF EQUATION 4 OF THE DISCUSSION . . . 47 C. INTEGRATION OF EQUATION 9 OF THE DISCUSSION . . . 50 REFERENCES . . . . . . . . . . . . . . . . . 55 LIST OF TABLES Table l. Kinetic Constants for K+ Activation of 5'AMP aminohydrolase at pHs 6.2, 6.5, and 6.8 . 2. Constants Used in Equation 4 of Discussion for Calculation of Theoretical Curves as Depicted in Figure 4 . . . . 3. Constants Used in Equation 10 of Discussion for Calculation of Theoretical Curves as Depicted in Figure 5 vi Page 14 30 36 LIST OF FIGURES Visible Absorption Spectrum of Resazurin Titration Curve of Resazurin . . . . Presentation of Change in Absorbance of Resazurin, Upon K+ Binding to 5'AMP aminohydrolase (1 mg ml' ) as per Equation 2 . . . Difference Titration Data and Theoretical Curves as Calculated from Equation 4 . . The pH Dependence of KA for K+ Activation of 5'AMP aminohydrolase and Theoretical Curves as Calcu- lated from Equation 10 . . . . . . . . . vii Page l5 lB 2T 28 34 5'AMP BSA EDTA MES Tris max LIST OF ABBREVIATIONS Adenosine 5'-Phosphate Bovine Serum Albumin Ethylenediaminetetraacetic Acid Concentration of substrate required for 50% saturation Concentration of activator required for 50% activation 2-(N-morpholino)Ethanesulfonic Acid Tris(hydroxylmethyl)Aminomethane Velocity at saturating concentrations of substrate viii INTRODUCTION The goal of this work was to examine the role of H+ in the KI mediated activation of 5'AMP aminohydrolase. Previous data (77) were consistent with the following activation scheme. K-a K-s KP E+AT+E'A+ST"+E'AS-—-+E’A+P a 4 S I ----—-- .0-- .....f where A is activator, S, substrate, P, product and E and E'A enzyme and activated enzyme, respectively. Activator A can take a variety of forms, e.g., 5'AMP, K+, ATP, ADP, and H+. In studying the H+ activation, both equilibrium and kinetic experiments will be performed. In addition, the relationship between the K+ and H+ activation processes will also be examined. LITERATURE REVIEW Occurrence of 5'AMP aminohydrolase 5'AMP aminohydrolase (E.C. 3.5.4.6) activity is found throughout the animal and plant kingdoms. Activity is present in rabbit (l-6), rat (8-12), mouse, guinea pig (8), calf (l3-lS), humans (20-23), cat, dog, (20), pigeon (8), chicken (31, 32, 35), duck. goose, turkey, turtle, horse, cow, goat, gerbil, hamster (37), frog, toad (33), snail (17), abalone (7), unfertilized fish eggs (24), elasmobranch fish (30), salmon, scallops, crab (34), pea seeds (29), and Aspergjllus oryzae (36, 37). Tissues possessing activity include skeletal muscle (3-6, 8, 9, ll), lung (9, 13), brain (l2, l3, 14), liver spleen, intestine, heart (9), erythrocyte (20-23), and human placenta (39). Within the cell the enzyme is located both in the structural and soluble fractions of the cell. In rat brain and liver (l2, 40), the enzyme is associated with the nuclear, microsomal, and mito- chondrial fractions of the cell. The rabbit muscle enzyme appears to be associated with myosin (4l). In heart tissue 5'AMP aminohydro- lase is located in the cytOplasm (42), or with mitochondrial, micro- somal, and nuclear cell fractions (43). In frog muscle the enzyme was found within the sarcolemna of the muscle fibers (l9). Erythro- cyles contain two forms of the enzyme, a soluble and a membrane- bound form. Approximately l5% of the total is of the latter type(32). 2 Purification 5'AMP aminohydrolase has been obtained in purified form from the following sources: rabbit muscle (5), pigeon muscle (8), chicken breast muscle (31), elasmobranch fish muscle (30), carp muscle (26), £5 gryzgg,(36), and calf duodenal (15). Purifications involved combinations of salt fractionizations, ion-exchange, and gel- filtration chromatographic steps. The procedure described by Smiley gt a1, (5) for rabbit muscle provides a simple one-step procedure, which has been adapted for use with other tissue sources (26, 30, 3l), making it a highly useful procedure. Structure The purified enzyme exhibits different molecular weight and quatenary structure depending upon the source. Rabbit muscle, chicken muscle, calf brain, and A. ggyggg.enzymes show multiple sub- unit structure. The rabbit muscle enzyme and the chicken muscle enzyme have four subunits which form a dimer of 560,000 molecular weight. The enzyme from A, ggyggg_is a dimer of 2l7,000 molecular weight with two subnits of 103,000 apiece (38). Characterization As a Metalloenzyme 5'AMP aminohydrolase from rat and rabbit muscle has been characterized as metalloenzymes having 2.0 (50) and 2.6 (5l) moles of zinc, respectively, per mole of enzyme. Enzyme from other sources is inactivated by chelating agents which is consistent with a metalloenzyme structure (46, 52). Activation and Inhibition 5'AMP aminohydrolase from many sources is activated by mono- valent cations. Cations shown to be effective are K+, Na+, Li+, NH+, Rb+, and Csf KI, Na+, and Li+ are usually the most effective as activators, but the relative order of effectiveness is dependent upon the source of enzyme. In each case, the monovalent cation decreases the Km for 5'AMP (8, 9, 22, 26, 40, 44-48). Metabolites such as ATP, GTP, GDP, and ADP modulate activity. These metabolites either activate or inhibit, depending upon source of enzyme, pH, and concentration of cation (8, 40, 44-48). Binding studies with ATP and GTP (6) and kinetic studies with substrate analogs (49) suggest that there are separate binding sites for 5'AMP and these metabolite activators. In general, anions inhibit the enzyme. These include F‘, I", Br', and Cl', with F' being the best inhibitor (8, 45, 47, 48). 'Chemical Modifications 1. Sulfhydryl Groups. The native rat muscle enzyme has 12 sulfhydryl groups which are accessible to reaction with DTNB or N-ethylmaleimide. These react without loss of enzyme activity (62). An additional 16 to 18 sulfhydryl groups react when zinc is removed. The latter are necessary for successful reconstitution of the enzyme with zinc (62). Some sulfhydryl groups of native rabbit muscle are involved in the binding of GTP. After treatment of the enzyme with 6.5 molar equivalents of p-mercuribenzoate, GTP binding was abolished (6). 2. Tyrosine. Through the use of l-fluoro-Z,4,-dinitroben- zene, arylation of the tyrosine residues was achieved. After short exposure to this reagent, the enzyme exhibited a higher Km for 5'AMP, with little decrease in maximum velocity. It was concluded that tyrosine residues are important in substrate binding (63). 3. Lysine. Six to seven lysine groups present in the rat muscle enzyme may be reacted with pyridoxal 5'-phosphate, and fixed by reduction with NaBH4. ATP and GTP protected against this reac- tion. It was suggested that the lysine residues are important for GTP and ATP binding (64). Physiological Role The physiological role of 5'AMP aminohydrolase at this time remains obscure. Several workers have postulated possible roles. These are listed below. 1. Catalyzing a reaction in the purine nucleotide cycle. fumarate adenylosuccinate 4 /—\GOP + PT aspartate Purine Nycleotide Cycle, after M. J. Lowenstein (1972), Physiological Review, 52, 382. This cycle allows, at least in muscle, the regulation of fumarate. 2. Another suggested role of 5'AMP aminohydrolase is the regulation of phosphofructokinase activity, an important control in glycolysis (60). The MHZ producued by the deamination reaction has been shown to activate phosphofructokinase (PFK). PFK has a KA of 0.33 m for NHZ. serve to raise the pH. PFK is inhibited by ATP at pH 7.1, but not Another aspect of this activation is that NH: could at pH 7.3. These combined effects would increase the activity of PFK (53). 3. 5'AMP aminohydrolase may be involved in the regulation of the relative concentrations of the adenine nucleotides (54). Under conditions simulating physiological, 5'AMP aminohydrolase was shown by Chapman and Atkinson (58) to be less active at an energy charge (E.C.) 0.9, than at lower values. This value is approximately that found in the liver, and other tissues (58, 59, 65). The depletion of ATP, during an energy-using cellular proc- ess, results in the concurrent decrease in the E.C. This activates 5'AMP aminohydrolase which converts AMP to IMP, increasing the mole fraction of ADP and ATP. The net result is the stabilization of the E.C., at lower adenine nucleotide concentrations. It was also noted (58) that the concentration of adenine nucleotides will not be lowered to a value which is not compatible with the normal function- ing of the cell. This lower limit is set because ATP is an activator for most 5'AMP aminohydrolases. When ATP concentration drops, the deamination of AMP will also decrease. Thus, 5'AMP aminohydrolase would function within the cell as a protection against drastic short- term decreases in the E.C. With the additional safeguard of not allowing complete depletion of the adenine nucleotides which would be necessary to maintain the E.C. 4. Muscular Dystrophy. The levels of 5'AMP aminohydrolase in skeletal muscle from human patients suffering from Duchenne muscu- lar dystrophy have been shown to be lower than in normal patients. This phenomenon was also observed in mice (61). Because of the important roles which can be postulated for 5'AMP aminohydrolase, as noted above, these low levels of activity could adversely affect muscle metabolism and may, in turn, contribute to the symptoms asso- ciated with Duchenne dystrophy. METHOD AND MATERIALS Enzyme Purification 5'AMP aminohydrolase (E.C. 3.5.4.6) was purified from mature rabbit back muscle in the manner of Smiley gt_§l, (5). All buffers and reagents were of the same composition as used by Smiley. Cellulose phosphate, used in the purification procedure, was washed with 10 volumes each of 0.5 N HCl, 0.5 M NaOH, and deionized water and then soaked several days in 10 mM Tris-EDTA before washing with deionized water. It was stored until use at 4°C. Just prior to use, the cellulose phosphate was equilibrated with extraction buffer. The purification was taken through to the cellulose phosphate step of the original procedure (5). The enzyme was then eluted with l.0 M KCl, 1 mM 2-mercaptoethanol, pH 7.0, instead of using a 0.45 to 1.0 M KCl gradient of the original procedure. Specific activities of between 80 and 130 units per mg of protein were obtained at 50 pM 5'AMP, pH 6.3. The enzyme was routinely stored at 4°C, in the presence of 1.0 M KCL, 1 mM 2-mercaptoethanol, pH 7.0, under a nitrogen atmo- sphere. There was negligible loss of activity after three weeks. In all experiments, enzyme was used within two weeks after preparation. Enzyme Assays 5'AMP aminohydrolase activity was determined using the spec- trophotometric assay of Kalckar (3). For 5'AMP concentrations above 1.0 mM the modifications by Smiley and Suelter (66) were used. The changes in optical density at 265 nm or at 285 nm were followed by the use of a Beckman DU spectrophotometer in conjunction with a Sargent-Welch recorder. Changes in optical density per minute were converted to umoles per minute using the conversion factors given by -l Smiley and Suelter (66) of 8.86 umoles ml.1 cm at 265 nm and 0.30 '1 at 285 nm. All assays were started by addition of umoles ml.1 cm enzyme. For routine assays the following buffer was used: 50 mM Tris-MES, 150 mM KCl, 50 BM 5'AMP, pH 6.3. For KA determinations the buffer used was 90 mM Tris-MES, 50 pM 5'AMP, 300 mM (CH3)4NC1, at the appropriate pH. One unit of enzyme activity is defined as umoles of 5'AMP deaminated per minute at 30°C. The Michaelis-Menten Kinetic parameters Km, KA, and Vmax were calculated after weighting the data to the reciprocal of the 4th power of the initial velocities as described by Wilkinson (68). This was accomplished with a computer using the program referred to as the Wilkin program. Removal of Activating Cations Activating cations were removed in either of the following two ways. For determination of K.A values where protein concentra- l tions below 0.5 mg ml' were suitable, a small volume of purified lO enzyme was passed over a column of Sephadex G-25 previously equili- brated with the buffer desired. The second procedure used for removing activating cations was by dialysis. Dialysis tubing (Union Carbide Corporation) was boiled in 2 mM sodium EDTA for one hour, rinsed twice with deionized water and stored in 2 mM sodium EDTA at 4°C. Before use all tubing was washed exhaustively with deionized water. Since it: was found that ionic strength was critical for enzyme stability, the following procedure was adopted. One ml of purified enzyme in 1.0 M KCl was dialyzed successively against two 500 m1 volumes of 0.5 M KCl and 0.3 M KCl, each for 12 hours. Each buffer also contained 1.0 mM Tris-MES at the appropriate pH and 1.0 mM 2-mercaptoethanol. Next the enzyme was dialyzed against two 500 ml volumes of 0.5 M (CH3)4NC1, 1 mM Tris-MES, each for 12 hours. The 0.5 M (CH3)4NC1 concentration was used instead of lower concen- trations because the (CH3)4N+ ion is noted to give a lower ionic strength than normally would be expected (69). Protein Determination Protein concentrations were determined by the tannic acid method of Katzenellenbogen and Dobryszyck (70) or by the method of Lowry (76) or by use of an extinction coefficient of 0.920 mg cm-1 ] as determined by Zielke (72). For the turbidometric method all ml- reagents were filtered before use. BSA in 1% NaCl solution was used as a standard. ll Proton Release and Uptake Experiments Protons released or absorbed by enzyme upon addition of K+ were detected in either of two ways. The first method involved the use of Resazurin. The second method involved the use of a hydrogen ion sensitive electrode (Sargent—Welch S-30070 combination electrode) in conjunction with a sensitive differential amplifier (Heath/Schlum- berger EU-200-30) coupled to a recorder (Heath/Schlumberger EU 205-1). For either method activating cations were removed from 5'AMP amino- hydrolase by extensive dialysis as described above. KA Determination Purified 5'AMP aminohydrolase was freed of activating cations by passing a small volume of enzyme over a Sephadex G-25 column according to the procedure described above. Initial velocities were determined as a function of K+ concentration using the assay pro- cedure described previously. An initial estimate of KA was determined by plotting the data after the fashion of Eadie-Hofstee (73). Here the residual activity of the enzyme in the absence of cation was subtracted from the initial velocity values at each cation concentration. More pre- cise values of KA were then determined, by using this initial esti- mate of KA to design kinetic experiments as suggested by Cleland (74). Thus, initial velocities in triplicate at 5 concentrations of cation between 20 and 80% saturation were determined. The points were then fitted to an Eadie-Hofstee plot, using the kinetic constants obtained from the Wilkin program (see Enzyme Assays, this section, for a description of this program). 12 As noted by Hemphill and Suelter (75), 5'AMP aminohydrolase was inactivated at high pH, giving anomalous kinetics. This inac- tivation is reduced as the monovalent cation, or protein concentra- tion is increased (75). Data when plotted as S/V versus 5 which showed a nonlinear response, especially at low cation concentrations, was discarded. In all cases, protein concentration in the assay was kept as high as possible. Reagents The Tris-Base, MES, 2-mercaptoethanol, and 5'AMP were obtained from Sigma Chemical Company (St. Louis, Missouri). The 5'AMP was either the free acid or the sodium salt. (CH3)4N01 and Resazurin were obtained from Eastman (Rochester, New York). The (CH3)4NC1 was recrystallized twice from isopropanol and stored at 110°C until use. Resazurin was of the certified type. The cellulose phosphate used in the purification was obtained from Brown Corporation (Berlin, New Hampshire). Its preparation for use is described in the section entitled Enzyme Purification. All other reagents used were of reagent grade or better. RESULTS Kinetic Constants for K+ Activation of 5'AMP aminohydrolase As a Function of pH The K+ activation constant along with Vma and the Hill slope, x n, were determined as a function of pH in order to establish the nature of the interaction of H+ and K+ with enzyme. Activating cations were removed from purified enzyme by gel-filtration, as described in Methods. Initial velocities were then measured at 30°C versus K+ ion concentration at 50 uM 5'AMP. The Wilkin program (see Methods for a description of this program) was used to analyze data for KA's and vmax' Values for n were determined from Hill plots. Values for these constants are presented in Table 1. These experi- ments demonstrate that the KA for K+ activation decreases with increasing H+ concentration, which suggests that KI and H+ are not competing for the same site. Maximum velocity and Hill slope under these conditions were independent of pH. These last observations are consistent with previous data (75). Release or Uptake of H+ Upon K+ Binding to 5'AMP aminohydrolase Resazurin, a proton sensitive dye, was used to examine uptake or release of H+ by enzyme upon addition of K+ to a solution of enzyme. Resazurin was first characterized as to its visible absorption spectrum and pK. Figure 1 presents the visible absorption spectrum of Resazurin at a concentration of 50 uM in 45 mM Tris-MES 13 14 TABLE l.--Kinetic Constants for K+ Activation of 5'AMP aminohydrolase at pHs 6.2, 6.5 and 6.8. pH KA (mM) v“) n(2) 6.2 1.96 t 0.16 96.8 r 13.9 0.98 i 0.03 6.5 3.46 t 0.25 109.0 i 20.0 1.01 t 0.08 6.8 6.10 x 0.26 113.5 E 12.0 1.02 e 0.01 Reaction mixtures contained 90 mM Tris-MES, 300 mM (CH NCl, and 50 DM 5'AMP. 3)4 1 (])Velocity (umoles min' mg'1) at saturating concentrations of KCl. (2)Hill slope as determined from plots of log (v/V-v) versus log (S). 15 my om.e eee .Amv om.m .Aqv ou.m .Amv ow.m .ANV o_.o .Apv om.m min mcwzoppom msp pm emcee -gmpwu mew; mmzlmwch 25 me newcwmpcou somezn m cm cm>Pommwc A2: omv :mc=~emmm mo ecuumam -ocpumam FN quoz zgmu < .umm: mm: moppm>=u Nunez: new: cwumeopoza .cwc=~emmm we Escuumam :omugcomn< anwmw>un.— acumen 16 A55 1523 m><3 own man .on a .2: .23 (km: tyou: will) 3 .8: .22: .OPXNF P 17 at various pHs. A maximum absorption at 600 nm was observed for the ionized form and 530 nm for the protonated form of dye. Titration of Resazurin with H+ produced data which fitted a theoretical curve, calculated from the Henderson-Hasselbalch equation, using a pK of 5.7 (Figure 2). Uptake or release of H+ upon K+ binding to enzyme, in the presence of Resazurin, was then examined by monitoring absorbance changes at 600 nm. Potassium was added to solutions containing 1.5 mg enzyme dissolved in one m1 of buffer composed of 1.0 mM Tris-MES, 300 mM (CH3)4NC1 and between 5.0 to 15 ug of Resazurin. Amount of dye added was dependent on pH of experiment because sensitivity of dye to changes in H+ ion concentrations is pH dependent. Experiments performed below approximately pH 6.15 shows an increase in absorbance indicating an uptake of H+ by enzyme. At higher pHs a decrease in absorbance was observed indicating a release of H+ by enzyme. Addi- tional experiments at pHs below pH 6.0 were attempted but due to the precipitation of enzyme accurate absorbance readings were not possible. Experiments above pH 7.0 were not performed because .earlier work demonstrated that enzyme at these pHs showed anomalous kinetics (75). As a control, K+ was added to buffer minus enzyme resulting in a decrease in absorbance at high cation concentrations, this being due to the effect of dilution. In experiments with enzyme present, sufficiently concentrated solutions of cations were added so as to eliminate this dilution effect. In addition, levels of enzymatic activity were measured before and after each experiment and were found to change less than 5%. 18 Figure 2.--Titration Curve of Resazurin. The fraction of change in absorbance, X+ at 600 nm was determined for a solution of 2.5 ug ml' . Resazurin dissolved in 100 mM Tris-MES as a function of pH. Solid line is a theoretical curve calculated using the Henderson-Hasselbalch equation with a pK of 5.7. N1 19 20 Data from these titration experiments were analyzed as follows. At zero cation concentrations, the absorbance at 600 nm was noted and was then subtracted from absorbance at 600 nm obtained after each addition of cation to give a net change in absorbance. Net changes in absorbances showed hyperbolic saturation with increas- ing K+ concentration and were found to fit equation 1 Mmax “(+1 KA + [K+] AA = (1) where AA is change in absorbance, and AAmax change in absorbance at saturating concentrations of K+. Equation 1 may be transformed into equation 2 giving a linear relationship between AA and K+ concentra- tion. Ll-RL— max max Likewise, equation 2 may also be transformed into an equation having the same functional form as the Lineweaver-Burk equation. Thus, the Wilkin program may be used to analyze data for KA's and AAmax‘ These constants were then used to plot data after equation 2 (Figure 3). Titration experiments were performed and analyzed in this manner over a pH range of 6.0 to 7.0. (The KA's are summarized in Figure 5, page 34). The slopes of plots shown in Figure 3 would normally be used to obtain information concerning the nature of interactions between two molecules for the same or different binding sites, 21 Figure 3.--Presentation of Change in Absorbance 0f Resazurin, Upon K+ Binding to 5'AMP aminohydrolase (1 mg ml") as per Equa- tion 2. Buffer included 1.0 mM Tris-MES, and 300 mM (CH3)4NC1. See text for explanation of data treatment. 22 pFl 6.8 4 _ pH 6.5 - 2 - 4 pH 6.1 pH 6.0 5 10 15 2'0 23 however, due to the varying sensitivity of Resazurin with pH, and absorption of H+ by byffer and enzyme, slopes at each pH cannot be compared. In order to overcome this, attempts were made to back- titrate the total change in absorbance so that the exact nature of these variables could be determined and the data corrected. Unfor- tunately, all attempts to back-titrate ended in precipitation of enzyme. This was probable due to locally high concentrations of base, in the cuvette, following addition of stock base solutions, prior to mixing. Lowering stock concentration of base to prevent this necessitated the addition of larger volumes of base which, in this case, was technically not possible due to volume limitations of cuvettes being used. It is important to note that these experiments were done in absence of substrate. Thus, K+ binds to free form of enzyme sug- gesting that the K+ mediated activation does not involve K+ binding to substrate or to the enzyme-substrate complex. This distinction becomes important when considering an activation scheme. Finally, binding of K+ to enzyme causes a release or an absorption of H+s depending on the pH of the solution. This would indicate that there are several types of functional groups present on the enzyme surface which are affected by K+ binding. Stoichiometry of H+ Released or Absorbed Upon KT Binding to 5'AMP aminohydrolase As a Function ofng A H+ sensitive glass electrode coupled to a differential amplifier (see Methods for a description of equipment used) was used 24 to determine the pH change following addition of K+ to a solution of enzyme. Here, back-titration could be performed without any of the problems associated with the Resazurin experiments. In these experiments the enzyme was freed of activating cations by dialysis, according to the procedure outlined in Methods. The pHs of solutions containing 1.5 mg/ml of enzyme in a buffer composed of 1.0 mM Tris- MES, and 300 mM (CH3)4NCl at the appropriate pH, were then monitored upon addition of K+. A control experiment was performed where pH of the above buffer solution, minus enzyme, was monitored upon addition of K+, resulting in no detectable change in pH. All experiments were performed under a nitrogen atmosphere to prevent absorption of CO by buffer. Data generated in this manner showed hyperbolic 2 saturation with increasing K+ concentration and were found to fit equation 3 + _ (Alemax [K ] ApH - + KA+EK1 where (ApH)max is the change in pH at saturating concentrations of K+. Equation 3 may be transformed into an equation having the same functional form as that of the Lineweaver-Burke equation. Thus, the Wilkin program may be used to analyze data for KA's and (ApH)max as an amount of OH' or H+. This was done by back-titrating pH changes. After saturating levels of K+ had been reached (100 x KA) several aliquots of (CH3)4N0H or HCl were added with pH change being noted after each addition. From such data standard curves of ApH - . + . versus OH or H concentrations were constructed. The slopes, 25 ApH/[OH'] or ApH/[H+] calculated from a least squares fit of data to a straight line, were then used, knowing volume of enzyme solution, to express (ApH)max as an amount of OH' or H+. The following formula was then employed to calculate numbers of H+ released or absorbed per molecule of enzyme: AV" = (B)(270,000) (4) H+ 1.5 x 10"3 where B represents amount of base or acid added to neutralize (ApH)max,270,000 the molecular weight of enzyme (6) and the factor of 1.5 x 10'3 grams represents amount of enzyme used in each experi- ment. These experiments were repeated over the pH range of 6.0 to 7.0 and Figure 4 (page 28) summarizes the data. DISCUSSION The goal of this work was to delineate the importance of H+ in the K+ mediated activation of 5'AMP aminohydrolase. A mechanism for K+ activation represented by the following scheme has been pro- posed by Suelter et_al, (77): K-a K«S KP E + A -——+-E'A + S -———+’E'AS -—-»-E'A + P Ka 1 KS i L _____________ 1 Scheme 1 where A is activator, S, substrate, P, product and E and E'A enzyme and activated enzyme, respectively. Activator, A, can take a variety of forms, e.g., 5'AMP, K+, ATP, ADP, and H+. Hydrogen ion is included because previous data (77) were consistent with such an activation. As results show, KA's for K+ activation generated by measuring H+ uptake or release using Resazurin and a Hydrogen ion electrode in the absence of substrate are simular to those obtained kinetically in presence of substrate (Figure 5). Thus, K+ activation of the enzyme can be portrayed by the first step in the above scheme. K a E + A 1:3:3-E'A (l) Ka 26 27 Changes in the equilibrium of equation 1 observed as a decrease in KA with decreasing pH implies a cooperative interaction between H+ and K+ binding sites. The stoichiometry of H+ binding can be estimated by fitting difference titration data to equation 2 (see Appendix A for the derivation of this equation) 2, na n _ i H T H “VF-El 2“? ‘2’ - H i H l where AVJL is the net difference in numbers of H+ released or absorbed by enzyme upon addition of saturating levels of K+ and Ki and KgA) are H+ ion dissociation constants in presence and absence of K+, respectively. Figure 4 shows difference titration data along with theoretical curves calculated from equation 2. It became apparent in early attempts to fit data that there are large numbers of groups undergoing pK changes. With so many variables (n1, n2, . ,(A (A A , . , , "2, k], k2, . . . , k£, k1 1, k2 ), . . . . kg ) ) it is impossible to obtain a unique fit to data and only the cases using the least numbers of variable constants are presented. This leads to a postulation of at least 18 H+ binding sites per subunit which are affected by K+ binding (Figure 4). A further conclusion which can be reached from these experiments is that the enzyme undergoes a conformational change upon K+ binding. Such a conclusion can be reached for the following reason. An anomalous pK for a group can result from hydrogen bonding (82), or from strong near-neighbor 28 Figure 4.--Difference Titration Data and Theoretical Curves as Cal- culated from Equation 4. Changes in pH, using equipment as described in Methods, of solutions containing 1.5 mg ml"1 5'AMP aminohydrolase dissolved in 1.0 mM Tris-MES, and 300 mM (CH3)4NC1 upon addition of KCl. All experi- ments were performed under an atmosphere of N2. The- oretical curves were calculated from equation 4 of Discussion for cases 1 (-——), 2 (---), 3 (---), and 4 (---) using constants given in Table 2. 29 00".“ [H"] x107 10 30 * TABLE 2.--Constants Used in Equation 4 of Disucssion for Calcula- tion of 'Theoretical Curves as Depicted in Figure 4. Case pK pK(A) 5.78 5.22 1 5.75 5.20 7.70 8.20 5.78 5.22 2 5.75 5.20 7.70 8.20 5.78 5.22 3 5.75 5.20 7.70 8.20 5.10 5.80 4 5.90 5.45 7.55 8.20 * See Discussion for explanation of constants. 31 electrostatic interactions (83) between this group and other nearby groups, or by the group itself being buried in a hydrophobic region (84). Thus, any conformational change will modify these types of effects producing a pK change. Now, by considering Wyman's theory of linked functions, KA versus pH data may be shown to satisfy a linkage equation (80), thus implying cooperative interactions between H+ and K+ binding sites. Hyman shows, from mass action considerations, that the basic linkage equation for binding of two ligands A and B is given by 80 317 a 1n a 3 ln a B A aA a where VA and VB are numbers of A and B ligands bound to enzyme, respectively, and aA and 8B are activities of A and 8 ions, respec- tively. A further linkage equation (which is suitable for integra- tion) may be derived from equation 3 and in our case for H+ and KI binding is given by —— (4) (see Appendix B for derivation of equation 4), where VK+ and VB are numbers of K+ are the sum of all other ions bound to enzyme, respec- tively, and aK+ is activity of K+ ion and aB is a composite activity of all other ions in equilibrium with enzyme. This equation must be 32 written in this form because there is no reason to suspect that K+ does not affect all types of ion equilibria. Given this, one can introduce the relationship a _ a (5) where a + and a - are activities of H+s absorbed and released by + .. enzyme, rexpectively, and aAl and a r are activities of buffer anions A2 which are in equilibrium with enzyme. Introduction equation 4 into equation 5 results in equation 6. 3V + aln a Bln a”: aln a 1 Din a .. __K__ = J + —_____ + _____A_.. J of 81n a _ aln a + aln a . 81n a . a V V V V-+ B+ K+ K+ K+ K (6) Experimentally, all H+s and all buffer ions in equilibrium with enzyme are indistinguishable, thus, equation 6 is rewritten as 8V aln a aln a + + - K —————fl—- + .____JE. (7) 3V3 a aln aK+ V aln aK+ V 8+ K+ K+ An equation similar to equation 7 has been derived by Wyman describ- ing the Bohr effect of Hemoglobin (80). In that case it was shown that the effect of anions on 02 binding was, within experimental 33 error, to be zero. If this approximation is used in our case, equation 7 becomes + + BVK 7 7 31 H (8) na H+a+ 1317+ H K which says that K+ binding is now solely dependent upon numbers of H+ bound to enzyme. Hyman (80) describes a procedure by which equation 8 can be integrated (integration is detailed in Appendix C) to give an equation showing KA as a function of aH+. This is given as a“+ K1 nI/q a + K2 nZ/q a + K2 n’L/q7 = n H H "A A —TA) —‘