Reaction mechanism of Ca2+-dependent adenosine triphosphatase of sarcoplasmic reticulum. ATP hydrolysis with CaATP as a substrate and role of divalent cation

ATP hydrolysis with CaATP as a substrate was characterized at 0 degrees C and pH 7.0 using purified ATPase preparations of sarcoplasmic reticulum and compared with that with MgATP as a substrate. The maximal rate of enzyme phosphorylation and the Km value for the phosphorylation were 8 to 10 times less for CaATP than for MgATP. Each substrate appeared to act as a competitive inhibitor with respect to the other in enzyme phosphorylation. The phosphoenzyme formed from CaATP turned over slowly because the conversion rate of the ADP-sensitive (E1P) to ADP-insensitive (E2P) phosphoenzyme was very slow. E2Ps, formed from both CaATP and MgATP, were similar in that KCl, MgCl2, or ATP accelerated their decomposition. Their sensitivity to KCl and/or ATP was retained even after a long incubation with excess EDTA. When the enzyme had been phosphorylated from CaATP, calcium remained bound to the enzyme even in the presence of excess EDTA. The observed parallelism between the amount and behavior of the enzyme-bound calcium and those of E2P strongly suggests that 1 mol of E2P has 1 mol of tightly bound calcium. During steady state ATP hydrolysis with CaATP as a substrate, a significant amount of the enzyme-ATP complex accumulated as a reaction intermediate because of slow dissociation of CaATP from the CaATP-enzyme complex and slow enzyme phosphorylation from the CaATP-enzyme complex. These results indicate that Mg2+ is not essential for the turnover of the calcium pump ATPase. It was proposed that the metal component of the substrate basically determines affinity of the substrate to the enzyme and the catalytic mechanism of subsequent reaction steps.     

Myosin motor domain lever arm rotation is coupled to ATP hydrolysis

We have investigated coupling of lever arm rotation to the ATP binding and hydrolysis steps for the myosin motor domain. In several current hypotheses of the mechanism of force production by muscle, the primary mechanical feature is the rotation of a lever arm that is a subdomain of the myosin motor domain. In these models, the lever arm rotates while the myosin motor domain is free, and then reverses the rotation to produce force while it is bound to actin. These mechanical steps are coupled to steps in the ATP hydrolysis cycle. Our hypothesis is that ATP hydrolysis induces lever arm rotation to produce a more compact motor domain that has stored mechanical energy. Our approach is to use transient electric birefringence techniques to measure changes in hydrodynamic size that result from lever arm rotation when various ligands are bound to isolated skeletal muscle myosin motor domain in solution. Results for ATP and CTP, which do support force production by muscle fibers, are compared to those of ATPgammaS and GTP, which do not. Measurements are also made of conformational changes when the motor domain is bound to NDP's and PP(i) in the absence and presence of the phosphate analogue orthovanadate, to determine the roles the nucleoside moieties of the nucleotides have on lever arm rotation. The results indicate that for the substrates investigated, rotation does not occur upon substrate binding, but is coupled to the NTP hydrolysis step. The data are consistent with a model in which only substrates that produce a motor domain-NDP-P(i) complex as the steady-state intermediate make the motor domain more compact, and only those substrates support force production.     

On the substrate specificity of the red cell calcium pump

ATP-dependent active calcium transport in inside-out human red cell membrane vesicles is stimulated by magnesium essentially parallel with an increase in MgATP concentration. At a constant, low (1 μM) calcium concentration, increasing ATP and magnesium increase the maximum calcium transport rate irrespective of the constant or decreasing concentrations of CaATP present. K_Ca for calcium pumping is practically unchanged at variable ATP and magnesium concentrations. Free magnesium above 1–2 mM inhibits active calcium transport, probably through a direct interaction with the transport enzyme. Based on the experimental findings reported we suggest that the true, physiological substrate of the red cell calcium pump is MgATP.     

Ultra-fastChara myosin: A test case for the swinging lever arm model for force production by myosin

Recent breakthroughs and technological improvements are rapidly generating evidence supporting the “swinging lever arm model” for force production by myosin. Unlike previous models, this model posits that the globular domain of the myosin motor binds to actin with a constant orientation during force generation. Movement of the neck domain of the motor is hypothesized to occur relative to the globular domain much like a lever arm. This intramolecular conformational change drives the movement of the bound actin. The swinging lever arm model is supported by or consistent with a large number of experimental data obtained with skeletal muscle or slime mold myosins, all of which move actin filaments at rates between 1 and 10 μm/sin vitro. Recently myosin was purified, fromChara internodal cells.In vitro the purifiedChara myosin moves actin filaments at rates one order of magnitude faster than the “fast” skeletal muscle myosin. While this ultra fast movement is not necessarily inconsistent with the swinging lever arm model, one or more specific facets of the motor must be altered in theChara motor in order to accommodate such rapid movement. These characteristics are experimentally testable, thus the ultra fast movement byChara myosin represents a powerful and compelling test of the swinging lever arm model.     

Myosin motors: missing structures and hidden springs

High-resolution structures of the motor domain of myosin II and lower resolution actin-myosin structures have led to the "swinging lever arm" model for myosin force generation. The available kinetic data are not all easily reconciled with this model and understanding the final details of the myosin motor mechanism must await actin-myosin co-crystals. The observation that myosin can populate multiple states in the absence of actin has nonetheless led to significant insights. The currently known myosin structures correspond to defined kinetic states that bind weakly (K(d)>microM) to actin. It is possible that the myosin lever arm could complete its swing before strong binding to actin and force generation--a process that would correspond, in the absence of load, to a Brownian ratchet. We further suggest that, under load, internal springs within the myosin head could decouple force generation and lever arm movement.     

Conformation of myosin interdomain interactions during contraction: deductions from muscle fibers using polarized fluorescence

Myosin cross-bridge subfragment 1 (S1) is the ATP catalyzing motor protein in muscle. It consists of three domains that catalyze ATP and bind actin (catalytic), conduct energy transduction (converter), and transport the load (lever arm). Force development during contraction is thought to result from rotary lever arm movement with the cross-bridge attached to actin. To elucidate cross-bridge structure during force development, two crystal structures of S1 were extrapolated to working "in solution" or oriented "in tissue" forms, using structure-sensitive optical spectroscopic signals from two extrinsic probes. The probes were located at two interfaces containing the catalytic, converter, and lever arm domains of S1. Observed signals included circular dichroism (CD) and absorption originating from S1 in solution in the presence and absence of actin and fluorescence polarization from cross-bridges in muscle fibers. Theoretical signals were calculated from S1 crystal structure models perturbed with lever arm movement from swiveling at three conserved glycines, 699, 703, and 710 (chicken skeletal myosin numbering). Best agreement between the computed and observed signals gave structures showing that actin binding to S1 causes movement of the lever arm. A three-state model of S1 conformation during contraction consists of three actin-bound cross-bridge states observed from muscle fibers in isometric contraction, in the presence of MgADP, and in rigor. Structures best representing these states show that most of the lever arm rotation occurs between isometric contraction and the MgADP states, i.e., during phosphate release. Smaller but significant lever arm rotation occurs with ADP dissociation. Structural changes within the S1 interfaces studied are discussed in the accompanying paper [Burghardt et al. (2001) Biochemistry 40, 4834-4843].     

MgATP binding changes neither the shape nor the flexibility of the heads of single myosin molecules.

The structural mechanism by which myosin heads exert force is unknown. One possibility is that the tight binding of the heads to actin drives them into a force-generating configuration. Another possibility is that the force-generating conformational change is inherent to the myosin heads. In this case the heads would make force by changing their shape according to the species of nucleotide in their active sites, the tight attachment to actin serving only to provide traction. To test this latter possibility, we used negative stain electron microscopy to search for a MgATP-induced shape change in the heads of single myosin molecules. We compared the heads of 10S smooth muscle myosin monomers (wherein MgATP is trapped at the active site) with the MgATP-free heads of 6S monomers. We found that to a resolution of about 2 nm, MgATP binding to the unrestrained myosin head does not drive it to change its shape or its flexibility. This result suggests that the head makes force by virtue of an induced fit to actin.     

Force generation by recombinant myosin heads trapped between two functionalized surfaces

Fluorescence resonance energy transfer measurements have revealed that the lever-arm domain of myosin swings when it hydrolyzes Mg-ATP. It is generally accepted that this swing of the lever arm of myosin is the molecular basis of force generation. On the other hand, the possibility that the force might be generated at the interface between actin and myosin cannot be ignored. However, there is a third possibility, namely, that myosin itself generates force without actin. Thus, using recombinant subfragment 1 molecules of Dictyostelium myosin II that were trapped between two functionalized surfaces of a surface-force apparatus, we determined whether myosin itself could actually generate force. Here, we report that, despite the absence of actin, myosin heads themselves have a capacity to generate a force (at least ~0.2 pN/molecule) that is coupled to the structural changes. Although the role of actin should not be neglected because muscle physiologically shortens as a result of the interaction between actin and myosin, in this work the focus is on the question of whether the catalytic domain of myosin has the capacity to generate force.     

Probing Muscle Myosin Motor Action: X-Ray (M3 and M6) Interference Measurements Report Motor Domain Not Lever Arm Movement

The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps. It was suggested that the sliding of myosin and actin filaments, the level of force produced and the lever arm angle are all directly coupled and that other models of lever arm movement will not fit the X-ray data. Here, we show that, in addition to interference across the A-band, which must be occurring, the observed meridional M3 and M6 X-ray intensity changes can all be explained very well by the changing diffraction effects during filament sliding caused by heads stereospecifically attached to actin moving axially relative to a population of detached or non-stereospecifically attached heads that remain fixed in position relative to the myosin filament backbone. Crucially, and contrary to previous interpretations, the X-ray interference results provide little direct information about the position of the myosin head lever arm; they are, in fact, reporting relative motor domain movements. The implications of the new interpretation are briefly assessed.     

Role of divalent cation bound to phosphoenzyme intermediate of sarcoplasmic reticulum ATPase

Effect of divalent cations bound to the phosphoenzyme intermediate of the ATPase of sarcoplasmic reticulum was investigated at 0 degree C and pH 7.0 using the purified ATPase preparations. Our previous study (Shigekawa, M., Wakabayashi, S., and Nakamura, H. (1983) J. Biol. Chem. 258, 14157-14161) indicated that 1 mol of the ADP-sensitive phosphoenzyme (E1P) formed from CaATP has 3 mol of high affinity binding sites for Ca2+, of which two are transport sites for calcium while the remainder is the acceptor site for calcium derived from the substrate, CaATP ("substrate site"). When incubated with a chelator of divalent cation, E1P formed from CaATP released all of its bound calcium to form a divalent cation-free phosphoenzyme. Evidence was presented that calcium dissociation from the substrate site was faster than that from the transport sites and primarily responsible for the ADP sensitivity loss of E1P induced by the chelator. Divalent cation-free phosphoenzyme was kinetically stable but when treated with divalent cations, it behaved similarly to the ADP-insensitive phosphoenzyme (E2P) which is the normal reaction intermediate of ATP hydrolysis. 45Ca bound at the substrate site on E1P formed from 45CaATP exchanged readily with nonradioactive ionized Ca2+ in the reaction medium whereas 45Ca at the transport sites on E1P was displaced only at a very slow rate which was almost the same as that for the phosphoenzyme hydrolysis. It was suggested that calcium at the transport sites on E1P formed from CaATP is released only after the rate-limiting conformational transition of the phosphoenzyme from E1P to E2P and that removal of calcium by a chelator from the substrate site facilitates this conformational transition, thereby allowing calcium bound at the transport sites to be released readily from the phosphoenzyme.     

Characterizations of cross-bridges in the presence of saturating concentrations of MgAMP-PNP in rabbit permeabilized psoas muscle.

Several earlier studies have led to different conclusions about the complex of myosin with MgAMP-PNP. It has been suggested that subfragment 1 of myosin (S1)-MgAMP-PNP forms an S1-MgADP-like state, an intermediate between the myosin S1-MgATP and myosin S1-MgADP states or a mixture of cross-bridge states. We suggest that the different states observed result from the failure to saturate S1 with MgAMP-PNP. At saturating MgAMP-PNP, the interaction of myosin S1 with actin is very similar to that which occurs in the presence of MgATP. 1) At 1 degrees C and 170 mM ionic strength the equatorial x-ray diffraction intensity ratio I11/I10 decreased with an increasing MgAMP-PNP concentration and leveled off by approximately 20 mM MgAMP-PNP. The resulting ratio was the same for MgATP-relaxed fibers. 2) The two dimensional x-ray diffraction patterns from MgATP-relaxed and MgAMP-PNP-relaxed bundles are similar. 3) The affinity of S1-MgAMP-PNP for the actin-tropomyosin-troponin complex in solution in the absence of free calcium is comparable with that of S1-MgATP. 4) In the presence of calcium, I11/I10 decreased toward the relaxed value with increasing MgAMP-PNP, signifying that the affinity between cross-bridge and actin is weakened by MgAMP-PNP. 5) The degree to which the equatorial intensity ratio decreases as the ionic strength increases is similar in MgAMP-PNP and MgATP. Therefore, results from both fiber and solution studies suggest that MgAMP-PNP acts as a non hydrolyzable MgATP analogue for myosin.     

High flexibility of the actomyosin crossbridge resides in skeletal muscle myosin subfragment-2 as demonstrated by a new single molecule assay

Popular views of force generation in muscle indicate that a lever arm in the myosin head initiates displacement of the thin filament. However, this lever arm is attached to the thick filament backbone by a flexible combination of coiled coils and hinges in the myosin subfragment-2 (S2); therefore, efficient force generation depends on tension development in this linking structure. Herein, a single molecule assay is developed to examine the flexibility of the intact S2 relative to that of the myosin head. Fluorescently labeled myosin rod is polymerized onto a single myosin molecule that is bound to actin, and the resulting Brownian motion of the rod is analyzed at video rates by digital image processing. Complete rotations of the rod suggest significant amounts of random coil in the linking structure. The close similarity of twist rates for double-headed and single-headed myosin indicates that most of the flexibility originates at or beyond the first pitch of coiled coil in S2 and most likely at the hinge connecting S2 and the light meromyosin. The myosin head has a smaller but still detectable impact on this flexibility, since the addition of ADP to the rigor crossbridge produces differential effects on the torsional characteristics of double-headed versus single-headed myosin.     

Calcium functionally uncouples the heads of myosin VI

This study examines the steady state activity and in vitro motility of single-headed (S1) and double-headed (HMM) myosin VI constructs within the context of two putative modes of regulation. Phosphorylation of threonine 406 does not alter either the rate of actin filament sliding or the maximal actin-activated ATPase rate of S1 or HMM constructs. Thus, we do not observe any regulation of myosin VI by phosphorylation within the motor domain. Interestingly, in the absence of calcium, the myosin VI HMM construct moves in an in vitro motility assay at a velocity that is twice that of S1 constructs, which may be indicative of movement that is not based on a "lever arm" mechanism. Increasing calcium above 10 microm slows both the rate of ADP release from S1 and HMM actomyosin VI and the rates of in vitro motility. Furthermore, high calcium concentrations appear to uncouple the two heads of myosin VI. Thus, phosphorylation and calcium are not on/off switches for myosin VI enzymatic activity, although calcium may alter the degree of processive movement for myosin VI-mediated cargo transport. Lastly, calmodulin mutants reveal that the calcium effect is dependent on calcium binding to the N-terminal lobe of calmodulin.     

X-ray structures of the apo and MgATP-bound states of Dictyostelium discoideum myosin motor domain

Myosin is the most comprehensively studied molecular motor that converts energy from the hydrolysis of MgATP into directed movement. Its motile cycle consists of a sequential series of interactions between myosin, actin, MgATP, and the products of hydrolysis, where the affinity of myosin for actin is modulated by the nature of the nucleotide bound in the active site. The first step in the contractile cycle occurs when ATP binds to actomyosin and releases myosin from the complex. We report here the structure of the motor domain of Dictyostelium discoideum myosin II both in its nucleotide-free state and complexed with MgATP. The structure with MgATP was obtained by soaking the crystals in substrate. These structures reveal that both the apo form and the MgATP complex are very similar to those previously seen with MgATPgammaS and MgAMP-PNP. Moreover, these structures are similar to that of chicken skeletal myosin subfragment-1. The crystallized protein is enzymatically active in solution, indicating that the conformation of myosin observed in chicken skeletal myosin subfragment-1 is unable to hydrolyze ATP and most likely represents the pre-hydrolysis structure for the myosin head that occurs after release from actin.     

Electron Tomography of Cryofixed,Isometrically Contracting Insect Flight Muscle Reveals Novel Actin-Myosin Interactions

Background

Isometric muscle contraction, where force is generated without muscle shortening, is a molecular traffic jam in which the number of actin-attached motors is maximized and all states of motor action are trapped with consequently high heterogeneity. This heterogeneity is a major limitation to deciphering myosin conformational changes in situ.

Methodology

We used multivariate data analysis to group repeat segments in electron tomograms of isometrically contracting insect flight muscle, mechanically monitored, rapidly frozen, freeze substituted, and thin sectioned. Improved resolution reveals the helical arrangement of F-actin subunits in the thin filament enabling an atomic model to be built into the thin filament density independent of the myosin. Actin-myosin attachments can now be assigned as weak or strong by their motor domain orientation relative to actin. Myosin attachments were quantified everywhere along the thin filament including troponin. Strong binding myosin attachments are found on only four F-actin subunits, the “target zone”, situated exactly midway between successive troponin complexes. They show an axial lever arm range of 77°/12.9 nm. The lever arm azimuthal range of strong binding attachments has a highly skewed, 127° range compared with X-ray crystallographic structures. Two types of weak actin attachments are described. One type, found exclusively in the target zone, appears to represent pre-working-stroke intermediates. The other, which contacts tropomyosin rather than actin, is positioned M-ward of the target zone, i.e. the position toward which thin filaments slide during shortening.

Conclusion

We present a model for the weak to strong transition in the myosin ATPase cycle that incorporates azimuthal movements of the motor domain on actin. Stress/strain in the S2 domain may explain azimuthal lever arm changes in the strong binding attachments. The results support previous conclusions that the weak attachments preceding force generation are very different from strong binding attachments.     

Oxygen-exchange studies on the pathways for magnesium adenosine 5'-triphosphate hydrolysis by actomyosin

At an intermediate stage in the hydrolysis of magnesium adenosine 5'-phosphate (MgATP) by myosin or actomyosin, there is an exchange of oxygen between water and the P gamma group of enzyme-bound nucleotide. Starting with [P gamma-18O]ATP as substrate, the exchange is revealed in the [18O]Pi species that are ultimately released as product into the reaction medium. An analysis of the distribution of these labeled Pi species, which contain 3, 2, 1, or none of the 18O atoms originally on the P gamma of ATP, is used to probe intermediate stages of the hydrolytic mechanism. In recent years, studies of this kind by several groups have shown that more than one pathway of hydrolysis operates. The work reported here demonstrates that two of these pathways are spurious; one is a "nonexchanging MgATPase" that is present in fresh myosin preparations; the other is an induced slow exchange that develops in myosin during storage (-20 degrees C) and subsequent aging (4 degrees C). However, after correction for these artifacts, two normal pathways for actomyosin hydrolysis remain. These normal pathways differ in the mode of interaction between actin and myosin in the course of hydrolysis; one is the Lymn-Taylor pathway where oxygen exchange occurs at a stage when actin and myosin are dissociated; the other is a pathway in which actin and myosin are associated during oxygen exchange. Each of these two pathways contributes an equal amount of Pi to the product pool. Thus, on average, each myosin head uses each of these pathways half the time. The findings suggest, e.g., that during contraction, myosin can dissociate from the actin filament only during every other cycle of MgATP hydrolysis or that only half the heads, at any one time, can exchange oxygen while free of the actin filament.     

Chemical Decoupling of ATPase Activation and Force Production from the Contractile Cycle in Myosin by Steric Hindrance of Lever-Arm Movement

The myosin motor protein generates force in muscle by hydrolyzing Adenosine 5′-triphosphate (ATP) while interacting transiently with actin. Structural evidence suggests the myosin globular head (subfragment 1 or S1) is articulated with semi-rigid catalytic and lever-arm domains joined by a flexible converter domain. According to the prevailing hypothesis for energy transduction, ATP binding and hydrolysis in the catalytic domain drives the relative movement of the lever arm. Actin binding and reversal of the lever-arm movement (power stroke) applies force to actin. These domains interface at the reactive lysine, Lys84, where trinitrophenylation (TNP-Lys84-S1) was observed in this work to block actin activation of myosin ATPase and in vitro sliding of actin over myosin. TNP-Lys84-S1's properties and interactions with actin were examined to determine how trinitrophenylation causes these effects. Weak and strong actin binding, the rate of mantADP release from actomyosin, and actomyosin dissociation by ATP were equivalent in TNP-Lys84-S1 and native S1. Molecular dynamics calculations indicate that lever-arm movement inhibition during ATP hydrolysis and the power stroke is caused by steric clashes between TNP and the converter or lever-arm domains. Together these findings suggest that TNP uncouples actin activation of myosin ATPase and the power stroke from other steps in the contraction cycle by inhibiting the converter and lever-arm domain movements.     

Myosin S2 Origins Track Evolution of Strong Binding on Actin by Azimuthal Rolling of Motor Domain

Myosin crystal structures have given rise to the swinging lever arm hypothesis, which predicts a large axial tilt of the lever arm domain during the actin-attached working stroke. Previous work imaging the working stroke in actively contracting, fast-frozen Lethocerus muscle confirmed the axial tilt; but strongly bound myosin heads also showed an unexpected azimuthal slew of the lever arm around the thin filament axis, which was not predicted from known crystal structures. We hypothesized that an azimuthal reorientation of the myosin motor domain on actin during the weak-binding to strong-binding transition could explain the lever arm slew provided that myosin’s α-helical coiled-coil subfragment 2 (S2) domain emerged from the thick filament backbone at a particular location. However, previous studies did not adequately resolve the S2 domain. Here we used electron tomography of rigor muscle swollen by low ionic strength to pull S2 clear of the thick filament backbone, thereby revealing the azimuth of its point of origin. The results show that the azimuth of S2 origins of those rigor myosin heads, bound to the actin target zone of actively contracting muscle, originate from a restricted region of the thick filament. This requires an azimuthal reorientation of the motor domain on actin during the weak to strong transition.     

The assignment of the Ca2+-ATPase activity of chromaffin granules to the proton translocating ATPase

CaATP is shown to function as a substrate for the proton translocating ATPase of chromaffin granule ghosts at concentrations which are comparable to that of MgATP. Using the initial rate of the proton pump activity as the measure (delta pH/delta t), an apparent Km-value of 139 +/- 8 microM was estimated for CaATP and 59 +/- 3 microM for MgATP. The maximal rate was markedly higher with MgATP than with CaATP, partly due to an inhibition of the hydrolytic activity at the higher concentrations of CaATP. The proton pump activity with CaATP was inhibited by N-ethylmaleimide and N,N'-dicyclohexylcarbodiimide at concentrations similar to that found for MgATP. No inhibition was observed with sodium vanadate in the concentration range 0-15 microM. Calmodulin and trifluoperazine had no effect on the overall ATPase activity with CaATP. These findings establish this activity as an intrinsic property of the chromaffin granules, i.e., linked to the H+-ATPase. No evidence was obtained for the presence of a Ca2+-translocating ATPase [Ca2+ + Mg2+)-ATPase) in the chromaffin granules.     

The role of tightly bound ADP on chloroplast ATPase

Isolated chloroplast coupling factor 1 ATPase is known to retain about 1 mol of tightly bound ADP/mol of enzyme. Some experimental results have given evidence that the bound ADP is at catalytic sites, but this view has not been supported by observations of a slow replacement of the bound ADP when CaATP or MgATP is added. The experiments reported in this paper show why a slow replacement of ADP bound at a catalytic site can occur. When coupling factor 1, labeled with tightly bound [3H]ADP, is exposed to Mg2+ or Ca2+ prior to the addition of MgATP or CaATP, a pronounced lag in the onset of ATP hydrolysis is observed, and only slow replacement of the [3H]ADP occurs. Mg2+ or Ca2+ can induce inhibition very rapidly, as if an inhibited form of the enzyme results whenever the enzyme with tightly bound ADP encounters Mg2+ or Ca2+ prior to ATP. The inhibited form can be slowly reactivated by incubation with EDTA, although some irreversible loss in activity is encountered. In contrast, when MgATP or CaATP is added to enzyme depleted of Mg2+ and Ca2+ by incubation with EDTA, a rapid onset of ATP hydrolysis occurs and most of the tightly bound [3H]ADP is released within a few seconds, as expected for binding at a catalytic site. The Mg2+-induced inhibition of both the ATPase activity and the lack of replacement of tightly bound [3H] ADP can be largely prevented by incubation with Pi under conditions favoring Pi addition to the site containing the tightly bound ADP. Our and other results can be explained if enzyme catalysis is greatly hindered when MgADP or CaADP without accompanying Pi is tightly bound at one of the three catalytic sites on the enzyme in a high affinity conformation.