ATP hydrolysis

THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed m U. S. A. Vol. 257, No. 20, Issue of October 25. pp. 12101-121(16, 1982 Mechanism of ATP Hydrolysis by Beef Heart Mitochondrial ATPase RATE ENHANCEMENTS RESULTING FROM COOPERATIVE INTERACTIONS BETWEEN MULTIPLE CATALYTIC SITES* (Received for publication, May 26, 1982) Richard L. Cross$, Charles Grubmeyerg, and Harvey S . Penefsky From the Department of Biochemistry, The Public Health Research Institute of the city of New York, h c . , New York, New York 10016 The very slow turnover rate for [y3’P]ATP hydroly- sis at a single catalytic site on soluble mitochondrial ATPase (F1) (uni-site catalysis) is accelerated over 10‘- fold when additional nonradioactive ATP is added to allow binding at multiple catalytic sites. This rate en- hancement increases turnover to V,, with no detect- able lag, thus demonstrating that the high affinity cat- alytic site previously characterized (Grubmeyer, C., Cross, R. L., and Penefsky, H. S . (1982) J. Biol. Chem 257, 12092-12100) is a normal catalytic site. When ATP binding at a second site is rate limiting for hydrolysis approximately 0.6 mol of ATP per mol of F1 is detected at catalytic sites. This level is predicted from the equilibrium distribution of substrate and products bound at a single site. The rate of ATP binding to a second catalytic site (6 X 10‘ M” s-’) is the same as the rate of binding at the first site. A K,,, = 30 PM and a V,, = 300 s-’ were measured under conditions that allow two sites to hydrolyze ATP. At higher ATP con- centrations an additional K, = 150 PM and V,, = 600 s-’ were measured suggesting the possibility of three functional catalytic sites on F1. The rate constants for the reaction mechanism de- veloped here and in the accompanying paper success- fully predict a lag time of about 10 ms in the early reaction kinetics of hydrolysis at 10 PM ATP. Hence, prior to addition of substrate F1 appears to be in a fully active form. The 10‘-fold rate enhancement obtained when sub- strate binds at a second catalytic site on the enzyme is accompanied by a 30-fold increase in the rate of ATP cleavage. However, the principal effect of substrate binding at the second site is a 10‘-fold increase in the rate of product release from the first site. These results provide a dramatic example of the potential magnitude of catalytic site interactions. The molecular mechanism of soluble mitochondrial ATPase is a problem of considerable importance to the role of the membrane-bound enzyme in ATP synthesis during oxidative phosphorylation. Under conditions for ATP hydrolysis that allow turnover at only a single site on the molecule (uni-site * This research was supported in part by Research Grants GM 21731 and GM 23152 from the National Institutes of Health, United States Public Health Service. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ On leave from the Department of Biochemistry, State University of New York, Upstate Medical Center, Syracuse, New York 13210. 0 Present address, Department of Biology, New York University, Washington Square East, New York, NY 10003. catalysis) it was shown (1) that substrate binds rapidly with very high affinity (K, = 1 O I 2 “I). Binding is followed by rapid development of an equilibrium between bound ATP and bound hydrolysis products, ADP and Pi. The equilibrium constant is 0.5. These observations support the concept that enzyme-bound ATP is formed from ADP and Pi with almost zero change in free energy and that the primary function of energy input during oxidative phosphorylation is to promote release of product ATP from catalytic sites on F1’ (reviewed in Ref. 2). It is clear from evidence currently available that the mul- tisubunit ATPase contains more than one catalytic site. Chemical modification studies and measurement of the num- ber of nucleotide-binding sites indicate that there are three copies of the p subunit (3, 4) and that this subunit contains the catalytic site (reviewed in Refs. 5 and 6). Direct evidence for the presence of at least two catalytic sites on F1 was provided by isotope-trap experiments with the ATP analog TNP-ATP (7). Strong cooperativity between catalytic sites also was demonstrable with both TNP-ATP and ATP. For example, hydrolysis of TNP-ATP bound in only one catalytic site was accelerated as much as 20-fold when a hydrolyzable nucleotide was made available to the second site (8). Studies of the I8O exchange between water and phosphate formed during hydrolysis of ATP also are compatible with the pres- ence of more than one catalytic site on the enzyme (9) and with the suggestion of catalytic site cooperativity (10). This paper undertakes an examination of the properties of catalysis that result when ATP is made available to multiple catalytic sites on the enzyme. Net turnover at a single site is enhanced over 106-fold when substrate binds to additional catalytic sites. These experiments also show that during steady state hydrolysis of ATP at low concentrations, signifi- cant and predictable levels of ATP are bound in catalytic sites. Successful predictions of presteady state kinetics and substrate modulation of the l8O exchange catalyzed by F1 are made using the rate constants provided in this and an accom- panying paper ( 1). MATERIALS AND METHODS ATP and CDTA were purchased from Sigma. 32Pi, enzyme grade, was obtained from ICN. [y3’P]ATP was prepared as described by Glynn and Chappell (11) with a specific activity in the range of lo5 to lo6 cpm/nmol. The radiochemical purity of ATP was determined by analytical thin layer chromatography as described (12). Adenine nucleotide concentrations were determined from the absorbance of solutions at 259 nm using a millimolar extinction coefficient of 15.4. Radioactivity was measured by scintillation counting (13). triphosphatase; TNP-ATP, 2’,3’-0-(2,4,6-trinitrophenyl)ATP; CDTA, ’ The abbreviations used are: F,, soluble mitochondrial adenosine trans-1,2-diaminocyclohexane-N,N,N’,N’-tetraacetic acid. 12101 12102 Modes of Catalysis by R-ATPase F, was isolated as described (14) and equilibrated on a centrifuge column (13) with Mg buffer or CDTA buffer. Mg buffer contained 0.25 M sucrose, 40 mM 4-morpholineethanesulfonic acid, 40 mM Tris, 1 mM KzHP04, and 0.5 mM MgS04, pH 8. CDTA buffer was identical with Mg buffer except that MgS04 was replaced by 2.5 m~ CDTA. Immediately after column centrifugation, MgS04 was added in 0.5 mM excess to CDTA. As shown earlier (1) the adenine nucleotide content of samples of Fl prepared in Mg buffer or in CDTA buffer was 3.5 or 2.8 mol/mol of F1, respectively. Quenched flow experiments were carried out in a Precision Syringe Ram, model UI-1001 (Update Instrument, Inc., Madison WI). The mixer and sampling valve configurations used in individual experi- ments are described in detail elsewhere (1). Experimental details are given in the figure legends. The isotope trap technique (15) was used to measure [y-”PIATP bound to catalytic sites on F1 in the experiments shown in Figs. 1 and 2. FI was first incubated with [Y-~~PIATP to form an F1. ”P complex, followed by addition of a large excess of nonradioactive ATP. Reaction was stopped by addition of perchloric acid, and 3>P, was measured as described below. The calculated and measured efficiency of the trap in preventing further binding of [Y-~~PIATP was excellent. Thus the only [y-3ZP]ATP that could hydrolyze during the cold chase was that which was already bound to the enzyme. This amount was determined by comparison to the ”Pi present in a sample that was acid quenched at the same time as the cold chase was initiated. The kinetic constants for ATP hydrolysis reported in Table I were measured in 1-ml reaction mixtures consisting of Mg buffer, 64 pg of pyruvate kinase, 5 mM phosphoenolpyruvate, 10 to 2000 ELM [y-”P] ATP (together with equimolar MgS04), and F,. The amount of F, added was sufficient to hydrolyze about 10% of the ATP in 25 s. The reaction was stopped by addition of 100 pl of 60% perchloric acid. At each concentration of ATP tested, a time course was constructed between 0 and 25 s in 5-s intervals. The linear portion of the time course was used to obtain the rate of hydrolysis. ”P, was determined by the method of Sugino and Miyoshi (16), using modifications introduced earlier (8) and with corrections for recovery of ”Pi. F1 protein was determined by a modified Lowry procedure (17) or a modified biuret procedure (18). A molecular weight of 347,000 (19) was used in all calculations. RESULTS In an accompanying paper, we show that ATP bound in a single very high affinity catalytic site on F1 (K , = 10” “I) is in rapid equilibrium with its bound hydrolysis products ADP and Pi (1). However, net hydrolysis of bound ATP, that is product release to form free enzyme, is very slow ( s-’ (1)). In contrast, the addition of excess nonradioactive ATP caused rapid hydrolysis and product release from the high affinity catalytic site, thus clearly distinguishing this site from the noncatalytic nucleotide-binding sites on the enzyme. In the present study, the ability of medium nucleotide to promote turnover of the high affinity catalytic site was used to deter- mine whether the site is a normal catalytic site capable of hydrolyzing ATP at the rapid steady state rates (600 s-’) observed in the presence of saturating concentrations of sub- strate. A complex was formed by incubating 0.15 PM [ y - 3 “ ] ATP with a 10-fold molar excess of F1 for 2 s in the quenched- flow apparatus. These are conditions for observing uni-site catalysis by F1 (1). The F1 -32P complex was then mixed in a second mixer (Fig. 1, zero time) with a large excess of nonra- dioactive MgATP (final concentration 3.3 m ~ ) . The reaction was allowed to proceed for periods of 5 to 40 ms before quenching by injection into perchloric acid. It may be seen that about 20% of the added [y-32P]ATP was hydrolyzed during the initial 2-s incubation (zero time). This amount of hydrolysis is consistent with rapid establishment of an equi- librium between bound substrate and products (1). It may also be seen in Fig. 1 that the bulk of the promoted hydrolysis was very rapid and occurred before 5 ms of chase time had elapsed. It is clear that at least 2 reaction half-times must have elapsed during the fist 5 ms giving a minimum rate constant of 300 s-’ for the fraction of ATP that hydrolyzes in t 20 4Y 2 = = h r 0.3 M ATP32 3 IO mM ATP 1 I H - I I I I I 1 0 5 IO 20 30 40 AGING TIME (mrec) FIG. 1. ATP-promoted hydrolysis of [y-‘”PIATP bound in a single site on FI. The experiment was carried out in a quenched- flow apparatus in the push-push mode and employing two mixers. Each of the 3 syringes contained 2.5 ml of Mg buffer with 10 mM P, and in addition: syringe 1, 3 p~ FI; syringe 2, 0.3 p~ [Y-~’P]ATP; syringe 3, 10 m~ MgATP. In the fwst push, equal volumes from syringes 1 and 2 were mixed in mixer 1 and allowed to age for 2 s in the length of hose designated “A” between mixers 1 and 2. During this time, the F1.32P complex was formed. The second push flushed the contents of hose A through the second mixer, where mixing with 10 mM nonradioactive ATP occurred. The reaction mixture then passed through a second aging hose into a vial containing 0.2 ml of 60% perchloric acid, 0.8 ml of Mg buffer, 0.1 ml of 100 m~ MgATP. The speed of the second push determined the aging time shown on the abscissa. This is the time elapsed between mixing the F1-”P complex with nonradioactive ATP and quenching in acid. The point representing zero aging time was obtained by disconnecting the sec- ond mixer and collecting the outflow of the first mixer directly in the perchloric acid quench. A 5-s time point (see text) was obtained by collecting the outflow of the second mixer in a vial containing Mg buffer and MgATP. Perchloric acid was then added 5 s later. A control experiment indicated that approximately 3 to 6% of the chase ATP was hydrolyzed during the 5-51 reaction. 32Pl formed was deter- mined as described under “Materials and Methods.” The data are expressed as per cent hydrolysis of the added [y3’P]ATP. this rapid phase. Since the turnover rate for net hydrolysis a t a single site on F1 is s” (I), the acceleration in rate caused by binding additional ATP is over 106-fold. Approximately 20% of the [y3’P]ATP initially added was hydrolyzed at a slower rate between 40 ms and 5 s (data not shown) indicating some heterogeneity in the FI . 32P complex. Since the biphasic nature of the ATP-promoted hydrolysis was influenced by P, and MgS04 concentration, the F1. 32P complex may be subject to ligand-induced asymmetries. About 13% of the added [ y - 32P]ATP did not bind at catalytic sites prior to addition of the nonradioactive ATP. In view of the substrate-product equilibrium at a single site ( K = 0.5, Ref. l), one might expect that during steady state hydrolysis, under conditions such that the rate-limiting step is the rate of binding of ATP to a second site, catalytic site occupancy by ATP would approach a value of 0.7. Fig. 2 shows an experiment in which the isotope trap technique (15) was used to measure [y-”’P]ATP bound to catalytic sites on the enzyme. At 20 n~ substrate and 2 n~ F,, a lag of about 2 s preceded the subsequent linear rate of hydrolysis (lower curue in Fig. 2 A , “acid quench”). The duration of the lag is consistent with the time required to build up a steady state level of enzyme-nucleotide complex. The difference between the ”Pi measured in the cold chase curve and in the acid quench curve is a direct measure of [Y-~’P]ATP bound to catalytic sites and committed to hydrolysis. The values for bound ATP are plotted in Fig. 2B. It may be seen that a steady state level of about 0.6 mol of [y3’P]ATP bound per mol of F1 was reached. Modes of Catalysis by Fl-ATPase 12103 3.01 - LL- 5 2 .0 - E ACID QUENCH 0 5 IO 15 20 REACTION TIME (sec) FIG. 2. ATP bound to catalytic sites on F1 during steady state hydrolysis at 20 n~ ATP. A, reaction mixtures contained, in a final volume of 1 ml , 20 IIM [Y-~’P]ATP in Mg buffer (pH 8.0). The reaction was started by addition of 2 pmol of Fl in 20 pl of buffer. At the indicated times the reaction was quenched by addition of 150 p1 of 60% perchloric acid, followed by 100 pl of 100 mM MgATP (0, m), or chased by addition of the MgATP first, followed 5 s later by the perchloric acid (0, 0). The presence of 10 pmol of nonradioactive ATP provided an effective isotope trap for [y-”PIATP not bound in catalytic sites since only 6 nmol of ATP was hydrolyzed during the 5- s cold chase. 32P1 was measured as described under “Materials and Methods.” Circles and squares represent 2 separate experiments. B, the difference between the cold chase and acid quench samples is plotted as a measure of [y-32P]ATP bound to catalytic sites on FI. The rate of hydrolysis of 20 I” ATP in Fig. 2 (0.16 mol of Pi/mol of Fl/s) is limited by the rate of binding of ATP to a second site on F1. This point is substantiated by the linear dependence of the rate of hydrolysis on the ATP concentra- tion at substrate levels between 5 and 60 I“ (Fig. 3 ) . The rate of ATP binding to a second site was determined from the slope to be 6.3 X lo6 M”.s”. This is the same as the rate of binding to the first site (I). The rate constants for the reaction mechanism developed previously (1) and in this paper were tested for their ability to predict the early reaction kinetics of ATP hydrolysis. The dashed line in Fig. 4 represents a computer-assisted numerical integration of the difference equation for Scheme 1. The rates of binding of ATP in steps 1 and 3 were taken to be equal (6 X lo6 M” . s-’, Fig. 3 and Ref. 1). The rate constants for the reversible hydrolysis of a single ATP on the enzyme (step 2) were 10 s-’ in the forward direction and 20 s-’ in the reverse (1). The rate of steps 4 plus 5 was taken to be 300 s-l as discussed below (Table I). It may be seen in Fig. 4 that the steady state rate at 10 PM ATP is the same for both the predicted and the experimental curves (about 50 turnovers per s). This rate is identical with one obtained in experiments done by hand under the same conditions but over longer time periods. The experimental time course (Fig. 4) shows a lag of about 10 ms. This is in good agreement with the predicted lag time of about 14 m s . A number of experiments of this kind were carried out at ATP concentrations in the range of 5 to 100 IJM. In all cases the lag time was less than 20 ms. In order to investigate further the hydrolysis of ATP at multiple catalytic sites on the enzyme, the substrate depend- ency of the reaction rate was measured over a concentration range from 10 to 2000 IJM ATP. The nonlinearity of the Lineweaver-Burk plots obtained from these measurements is in agreement with the apparent negative cooperativity re- ported by Ebel and Lardy (20). The K,,, and VmaX values calculated from the linear portions of the plots are shown in Table I. For purposes of comparison the Kd and turnover rate for hydrolysis of ATP in a single site (1) are shown in line 1 of the table. The turnover rate of uni-site catalysis is too slow 0.4 I 0 2 0 40 60 ATP CONCENTRATION (nM) FIG. 3. Rate of binding of ATP at a second site on F1. The reaction mixtures contained 0.25 to 3 pmol of F1 and a 20-fold molar excess of [Y-~’P]ATP in 1 ml of Mg buffer. The rate of hydrolysis was measured by acid quenching seven samples at various times over a period sufficient to give about 2.5 turnovers (8 to 80 s). 32P, was measured as described (see under “Materials and Methods”). The turnover rate is plotted versus the ATP concentration. 8 - - LL- 1 0 50 100 150 20( AGING TIME (rnsec) FIG. 4. Early reaction kinetics of ATP hydrolysis by F1. The experiment was carried out in a quenched flow apparatus. The single mixer configuration was employed with (0) and without (X) the sampling valve described (see under “Materials and Methods”). Both syringes (A and B) contained 2.5 ml of Mg buffer, pH 8, and in addition, A, 0.5 p~ FI; B, 20 p~ [y-32P]ATP. The velocity of the single push controlled the aging time shown on the abscissa. The outflow of the aging hose was collected in vials containing 0.15 ml of 60% perchloric acid and 1 ml of Mg buffer. At the end of the experiment, 5O-pl samples were taken from syringe B to measure 32P, at zero time. 32P, was determined as described (see under “Materials and Meth- ods”). -, drawn through experimental points; - - -, represents a computer-assisted numerical integration of the difference equation for Scheme 1 as described in the text. ATP -2112 ATP 1 4 ADP+ Pi .ADP+i\ .ADP.Pi 1 ~ FI 3b !‘ATP ‘‘ATP F F SCHEME 1 12104 Modes of Catalysis by K-ATPase TABLE I Kinetic constants for ATP hydrolysis at multiple catalytic sites Mode of catalysis K d or K , V,, M S-1 Uni-site lo"* Bi-site" 3 X 300 Tri-site" 1.5 X 600 a Reaction kinetics were measured as described under "Materials and Methods." The rates were analyzed usi