Whether two or three catalytic sites per enzyme were present was not known at that time.
Advances in Enzyme Regulation, Volume 42 - 1st Edition
We proposed alternating behavior of two sites, although it was recognized that the results would also be compatible with sequential participation of three sites 53 , During net ATP formation or hydrolysis, sites were considered to proceed sequentially through the steps of binding, interconversion of reactants, and release so that at any one time each catalytic site was at a different stage of the catalysis.
The concept seemed attractive, but more evaluation was needed. David Hackney, a talented postdoctoral fellow from Dan Koshland's laboratory, had joined our group and initiated his excellent experimental and theoretical studies of the oxygen exchanges. If dynamic reversal of ATP formation at a catalytic site continued in the absence of net reaction, then reductions in the concentration of P i or ADP should increase the amount of intermediate oxygen exchange per ATP made.
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We were encouraged by a report from a former postdoctoral fellow of our group, Robert Mitchell, that he and his colleagues observed increased intermediate oxygen exchange accompanying ATP hydrolysis by submitochondrial particles when ATP concentration was lowered Support for the possibility also came from the observation of Wimmer and Rose 56 that when ATP was exposed to chloroplasts in the light, the ATP showed nearly complete exchange of its oxygens before being released.
This is as expected if low ADP concentration in the medium prevented the release of the ATP and many reversals occurred before its release. Hackney observed that during net oxidative phosphorylation as either ADP or P i concentration was decreased, there was a marked increase in water oxygen incorporation into each ATP formed Additional observations made it unlikely that some type of enzyme heterogeneity or hysteresis could explain the exchange patterns.
It deserves emphasis that these experiments were performed with submitochondrial particles during net ATP synthesis, giving them relevance to the actual oxidative phosphorylation process. An interesting possibility was that catalytic site cooperativity might also be found with the isolated F 1 -ATPase. We tested this at millimolar concentrations of ATP and found that the P i formed contained only close to the one water oxygen necessary for the hydrolysis.
This was found to be so 58 and as ATP concentrations were lowered the number of reversals before the P i was released approached a limit of over The reaction rates and equilibrium characterizing the slow catalysis at a single site were determined in a widely recognized study by Cross together with Grubmeyer and Penefsky In a slow reaction, needing relatively high P i concentration, a tightly bound ADP became phosphorylated.
Other findings made it probable that this was at the same site as the ADP that was rapidly released in the acid-base transition of thylakoid membranes and thus that this site was likely where covalent bond formation occurred during photophosphorylation. In addition, results of various investigators established that chemical modification of only one catalytic site effectively stopped catalysis and that each of the three catalytic sites had a different capacity for derivatization.
Such behavior agreed with the concept that during catalysis all three catalytic sites were in different conformations and proceeded sequentially through the conformations. As covered in the Appendix of a review there is strong support for this interpretation This includes demonstrations that the P i oxygen exchanges catalyzed by the sarcoplasmic reticulum ATPase 64 , 65 and pyrophosphatase 66 , 67 , as well as that of myosin ATPase as mentioned above, result from reversible formation of a phosphorylated enzyme or enzyme-bound pyrophosphate or ATP, respectively.
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Other evaluations of our postulates were needed. Rapid mixing and quenching techniques yielded essential information. We used rapid mixing in an acid-base transition of chloroplast thylakoid membranes, as introduced by Jagendorf and colleagues, to start ATP synthesis in a few milliseconds.
As substantiated in later experiments, the tightly bound ADP in such chloroplast membranes prior to release is tightly bound at a catalytic site without P i. The demonstration that exposure to protonmotive force caused the release of a tightly bound ADP from a catalytic site without phosphorylation had important implications for later developments.
Thus such inhibition in the intact synthase is readily and quickly overcome by protonmotive force. Our rapid mixing experiments verified that medium ADP was rapidly bound and phosphorylated as if no phosphorylated intermediates were involved. They provided evidence that during photophosphorylation, in addition to a transitorily bound ATP, about one bound P i and one bound ADP per enzyme are present and committed to ATP synthesis Such results harmonize with the alternating site model with more than one catalytic site having bound reactants, as required if a tight site is already filled and substrates must initially bind at another site.
Research conferences are important to scientific progress because concepts can be freely discussed, and the publication of proceedings often allows inclusion of material not suited for the usual journals. For example, in my contribution to a conference honoring Ef Racker, I summarized our concepts and considered how to name our suggested mechanism. A name seemed desirable for ease of discussion and to identify the concept in the field.
The binding change mechanism at that time included the following concepts. The first compound made from P i is ATP itself no intermediates ; a principal requirement of energy is not for the formation but for the release of ATP; energy input also promotes the competent binding of P i and the sequential participation of catalytic sites so that binding of substrate s at one site is necessary for release of product s from another site.
Two years later, another and even more novel concept of the binding change mechanism was developed, namely the proposal of rotational catalysis.
The suggestion that rotation of internal subunit s drives the binding changes for catalysis was first published in reports from and conferences at the University of Wisconsin 72 , How this concept came about is outlined next. In the s highly enriched 18 O was available, mass spectrometry techniques for 18 O analysis had improved, and Mildred Cohn had introduced an NMR method for measuring 18 O in phosphate compounds.
David Hackney developed theoretical aspects of 18 O measurements relevant to observed distributions of 18 O isotopomers of P i with 0 to 4 18 O atoms per P i or 0 to 3 18 O atoms per ATP molecule. More importantly, the distribution of 18 O isotopomers corresponded to that statistically expected if all the ATP were produced by the same catalytic pathway.
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This eliminated the possibility that substrate modulation arose from heterogeneity of the enzyme used and made modulation by control sites unlikely. We now regarded the catalytic site cooperativity of ATP synthase to be reasonably well established. At appropriate labeling and substrate concentration ranges, the distribution patterns provided a sensitive test for more than one catalytic pathway. A statistically homogeneous distribution meant that every substrate that reacted faced the same possibilities of proceeding through the same reaction steps.
This means that rate constants governing the binding and release of substrate s , their reversible interconversion, and the release of product were the same. To me, the power of this type of 18 O use is unusual and indeed a bit awesome. Catalytic sites on multisubunit enzymes can be very sensitive to conformational changes in adjacent subunits. The evidence that all three sites conducted catalysis identically was compelling to me. The more I puzzled about these aspects, the more it seemed that there was only one satisfactory answer.
When I first presented this concept to my research group, their acceptance was initially quite reserved they knew all too well that I could be wrong.
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With further consideration, they became interested and supportive. Much remained to be explored, and some experimental approaches are summarized in the next few sections. The chloroplast F 1 -ATPase showed a similar behavior, and the distribution of the 18 O isotopomers in the P i formed corresponded to a single catalytic pathway Various wild type and mutant E. However, the distribution of 18 O isotopomers with the E. A question had been raised about whether the F 1 -ATPase from a thermophile showed catalytic cooperativity because uni-site catalysis was not readily apparent.
A cooperative experiment disclosed the expected modulation of the oxygen exchange but at a higher range of ATP concentration These various results meant that the increase in the extent of oxygen exchange with each P i formed which occurs with a decrease in the ATP concentration is likely a general property of all F 1 -ATPases and supports the probability that all ATP synthases share a common mechanism.
We felt that it should show similar oxygen exchange properties, and measurements demonstrated that this was so We devised methods to measure bound reactants during steady-state ATP synthesis. A hexokinase accessibility method gave a measure of bound ATP, and a rapid dilution of medium 32 P i gave a measure of bound P i committed to form ATP.
Measurements during photophosphorylation showed that even at lower substrate concentrations the total of catalytic site-bound ATP and committed P i was greater than one per enzyme, as anticipated if the proposed catalytic site cooperativity was occurring. During photophosphorylation, 32 P i rapidly labeled catalytic ATP, and then the medium [ 32 P]ATP formed was incorporated much more slowly into the non-catalytic sites When illumination ceased, the catalytic site ATP continued to show 18 O exchange 81 , meaning that reversible formation of bound ADP was still occurring.
Within minutes the P i dropped off, leaving a tightly bound ADP at the catalytic site Such results helped explain labeling patterns we and others had observed and supported our concepts of tightly bound ATP as an intermediate and of catalytic site cooperativity. Occasionally in biochemical research one encounters a property of a system that seems designed to confuse and thwart the researcher. Clarification of this unusual role of a tight ADP was necessary for an adequate understanding of the proposed binding change mechanism.
Hackney noted the inhibition was slowly reversible by ATP addition Observations in Vinogradov's laboratory showed that the inhibition depended on the presence of tightly bound ADP and that the Mg-ADP-inhibited form was stabilized by azide Subsequent studies in our and other laboratories revealed characteristics of the inhibition. Added ATP promotes slow release of the inhibitory ADP from a catalytic site as an increase to a steady-state rate is attained. At steady state, a slow interconversion of active and inactive forms continues. P i and various anions activate by promoting release of the ADP.
The inhibitory ADP is at a catalytic site, not at a regulatory site as had been suggested. Another important result of our continued probing was the recognition that, under some conditions, the presence of ATP at a certain non-catalytic site is necessary for the onset of activity of the chloroplast F 1 -ATPase This was the first recognized function for a non-catalytic bound ATP. The action was found to result from acceleration of the release of the inhibitory ADP from catalytic sites that follows the addition of medium ATP From the above it is apparent that complicated rate patterns may be found.
It is probable that with all F 1 -ATPases, and even under favorable conditions, a fair portion of the enzyme may be in the inhibited form. Many reported and planned experiments may be undermined by an unrecognized occurrence of the Mg-ADP inhibition. A procedure for estimating the portion of the enzyme in the inhibited form, as developed by Murataliev 87 , deserves wider application. This is akin to the removal of inhibitory imido-ATP that blocks hydrolysis but not synthesis.
By the mids, the sequence of the ATP synthase subunits was becoming available. An ATP derivative, 2-azido-ATP, which serves as a good substrate and upon photolysis becomes covalently attached, was described in Lardy's laboratory. The sites were thus near subunit interfaces. Sites with similar conserved sequences were noted with the mitochondrial, chloroplast, and E.
Whether the liver 93 and chloroplast enzymes see Ref.