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Home » Biology Articles » Biophysics » Medical Biophysics » The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease » Consistency of Current Mechanisms of ATP Synthesis and Muscle Contraction with KeyExperimental Data

Consistency of Current Mechanisms of ATP Synthesis and Muscle Contraction with KeyExperimental Data
- The New Unified Theory of ATP Synthesis/Hydrolysis and Muscle Contraction, Its Manifold Fundamental Consequences and Mechanistic Implications and Its Applications in Health and Disease

3.1. Bioenergetics

3.1.1. Nucleotide Binding Affinity (Kd) Measurements at the Catalytic Sites of F1-ATPase

As discussed in Sections 2.3 and 2.5.2, the binding change mechanism was based on the tenet that the ATP synthase can stabilize the MgATP differentially with respect to MgADP and Pi by binding the MgATP very tightly (e.g. in a highly sequestered site where the reaction is at equilibrium with Keq~1, and there is free dynamic reversal on the enzyme surface), and that energy input occurred not at the bond formation step during ATP synthesis but at the step of product MgATP release [7]. This can be verified by evaluating measurements of nucleotide binding affinity to the catalytic sites of F1-ATPase. Earlier measurements of the binding affinity of MgATP to site 1 in mitochondrial F1 indicated a Kd1 value of ≤ 1 pM [45]. Since MgADP was taken to bind with a Kd2 of ~10 μM in site 2, a difference of binding energy ≥ RT ln (Kd2 / Kd1), i.e., ~35 kJ/mol was inferred. However, as reviewed by us previously [1, 2], very careful measurements of binding affinities over a decade of experimentation through the development of a true equilibrium technique using optical probes that could directly monitor catalytic site occupancy, Senior and colleagues have proved that the MgATP binds to site 1 of E. coli F1 with a Kd of only 0.02 μM (and not 1 pM), and to site 2 with a Kd of ~1 μM, and that MgADP binds to site 2 with a Kd of ~25 μM [1-3]. Assuming additivity of binding energies in site 1 and site 2, calculation reveals that the binding energy of phosphate in its site is ~7-10 kJ/mol, and that the binding energy difference between MgATP in site 1 and MgADP.Pi in site 2 is only ~7-10 kJ/mol (and not the ≥ 35 kJ/mol postulated in the binding change mechanism). Thus this postulate of the binding change mechanism is not consistent with the biochemical nucleotide binding affinity (Kd) experimental measurements, which are now generally accepted. It should be noted that there is no difficulty for the torsional mechanism, which is consistent with these experiments. The sequential ~9 kJ/mol + ~9 kJ/mol released upon ATP hydrolysis (Section 2.4.3) are immediately used to weaken phosphate binding and weaken a subunit-subunit interaction and lead to no net free energy changes, while the ~18 kJ/mol of the hydrolysis free energy is kept stored as potential energy and is released at a later step upon phosphate release, causing rotation of a subunit, or is stored in a localized region of the macromolecule for performance of useful work subsequently upon release of the stored energy, as discussed in Sections 2.4.2, 2.4.3, 2.5.1 and 2.5.2, again leading to a net change of zero free energy. The concomitant increase of binding energy of MgATP in site 1 by ~9 kJ/mol (over MgADP.Pi in site 2) during physiological ATP synthesis during the L to T transition (Section 2.4.1), and the concomitant reduction of MgADP binding in site 2 by ~9 kJ/mol during the bond cleavage step in hydrolysis during the T to L transition (Section 2.4.3) is consistent with the nucleotide binding affinity measurements on F1-ATPase, and the binding energy difference of ~7-10 kJ/mol above. Finally, it should also be pointed out that the postulate of a highly sequestered site binding MgATP (or an ATP analog) with a Kd of 1 pM or lower is not consistent with recent experimental data which show that the Kd for MgITP binding in the catalytic site with the highest affinity of E. coli F1 is only 0.33 μM, yet MgITP is readily synthesized by E. coli ATP synthase [3]. In summary, it may be stated that the binding change mechanism is not consistent with basic biochemical data on nucleotide binding affinities at the catalytic sites of F1-ATPase and hence, in addition to the plethora of reasons given in this paper and in earlier papers [1, 2, 10, 11, 13, 16-18], it is absolutely clear that the mechanism requires revision.

3.1.2. Structure of Mitochondrial F1-ATPase with Nucleotide Bound to All Three Catalytic Sites

It has already been reviewed how the F1-ATPase structure containing nucleotide bound to all three catalytic sites is consistent with the torsional mechanism, but completely inconsistent with the binding change mechanism (p. 73 of Ref.[1]). It should also be mentioned that upon superposition of the more recent structure above with the original native mitochondrial F1 structure, it was stated in 2001 (p. 338 of Ref. [33]) that (in the two structures) “the two conformations of the γ-subunit are related by a rotation that varies in magnitude along the length of the C-terminal helix, ranging from less than 1o for the final residues (γ 259-272) to a maximum of about 20o for residues γ 234-244 (which form the coiled coil with residues γ 20-10).” Further, it was stated that “the variation in the rotation angle implies that the coiled-coil is slightly more twisted in the (ADP.AlF4 -)2F1 structure than in the native form,” and further on that “the rotation is not uniform over the length of the γ-subunit.” These were important observations being made for the first time, thanks to the possibility of comparison afforded between two γ conformations due to the solution of the second key structure. However, all the longer statements within quotes could be considerably condensed by calling it “torsional strain” in the γ- subunit, as predicted by the torsional mechanism from inception. This central characteristic led to the title of one of our earlier papers [18] and the naming of the mechanism as “the torsional mechanism” from 1999, and subsequently as “the torsional mechanism of energy transduction and ATP synthesis”. In fact, the experimental observations made for the first time in 2001 [33], and inserted within quotes here, define, (and are the very inherent attributes of) torsion, and thus offer further important evidence in support of the torsional mechanism. It should however be added that, in contrast, the rotation of the γ-subunit upon “substrate binding” (or rather, ADP-ATP exchange), and “product release” (i.e., Pi release) steps is not “small,” as postulated [33], but as large as ~80o and ~40o respectively, as evident from the unified quantum theory of protein molecular machines synthesizing/hydrolyzing ATP developed in this paper. In summary, the torsional mechanism is in agreement with all the structural observations. We believe that interpretation of structural, biochemical and biophysical data in the light of the torsional mechanism (as opposed to the binding change mechanism) will help solve the longstanding difficulties in the field and also lead to considerable and rapid progress in the near future.

3.2. Muscle Contraction

3.2.1. Contraction Characteristics and ATPase Activity of Muscle Fibers in the Presence of Antibody to Myosin S-2

In an interesting experiment, Sugi, Harrington, and colleagues examined the contraction characteristics and MgATPase activity of glycerinated muscle fibers prepared from rabbit psoas in the presence and absence of polyclonal antibody directed against the S-2 region of myosin [44]. Their results indicated that Ca2+-activated force and muscle fiber stiffness were uncoupled from MgATPase activity in the antibody-treated muscle fibers. In other words, when anti-S-2 antibody attaches to the S- 2 coiled coil of myosin molecules, their heads still hydrolyze ATP, but no longer contribute to force production and muscle fiber stiffness. This experimental fact cannot be explained by previous mechanisms of muscle contraction, including the lever arm model, which do not postulate any role for the S-2 coiled coil in the generation of elementary contractile force by myosin. The question that arises, which is unanswered by previous mechanisms and biochemical/ mechanochemical schemes, is how antibody attached to the S-2 coiled coil of a myosin molecule could exert a long-range effect on the mechanochemical properties of the myosin heads? This question is readily answered by the rotation-uncoiling-tilt (RUT) energy storage mechanism of muscle contraction, in which a transmission of the free energy release of MgATP binding, hydrolysis and phosphate release by the physicochemical processes occurring in the myosin head takes place all the way to S-2 via the helical

structure of myosin and the S-1–S-2 hinge (i.e. head-rod junction), stores the free energy in a highenergy uncoiled state of the S-2 coiled coil, which is then released back from S-2 to S-1 and the actomyosin contacts via the S-1–S-2 hinge after the myosin heads have bound tightly to actin sites, and causes a powerstroke of the myosin heads bound to actin with the S-1–S-2 hinge as fulcrum. Thus, the RUT energy storage mechanism of muscle contraction can explain all the experimental facts usually given in support of the lever arm model, and also other longstanding experimental facts that have proved very hard to explain by other mechanisms of muscle contraction. Sugi and Harrington [44] concluded that the myosin hinge plays an essential role in muscle contraction, and that much more attention must be paid to the function of this region. To this we would like to add that the S-2 coiled coil also plays an essential role during muscle contraction by storing the free energy of the MgATPase processes occurring in the myosin head, and recommend that the S-2 region also needs to be paid greater attention by the muscle contraction and motility community, and that the RUT energy storage mechanism of muscle contraction can serve as a basis for further experimentation in this field.

3.2.2. Single Molecule Imaging

Several single-molecule imaging studies in the field of motility have been carried out in the last ten years, though most of them have employed unconventional myosins (such as myosin V and myosin VI), or kinesins, or other processive molecular motors as model systems (Section 6), partly because of their larger step size (often tens of nanometers), compared to myosin II, and their processive character. A powerful single-molecule study that deals with muscle myosin as the experimental system is that by Yanagida and his coworkers [45], where the chemical ATPase and mechanical events were simultaneously measured in single myosin molecules during force generation and interaction with actin. The experiment reveals unambiguously that myosin II can produce force several hundred milliseconds after MgATP hydrolysis and release of bound nucleotides. This important finding [45] does not support several existing models in which force production is directly coupled to the release of bound ligands. The delay in force generation after release of bound ligands from the catalytic site in myosin S-1 points to the ability of myosin II to store chemical energy from ATP binding, hydrolysis and product release and subsequently perform useful work with the stored energy, as postulated in a central tenet of the RUT energy storage mechanism of muscle contraction [2, 10, 11, 14].

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