Biomedical subjects
Publications and source records attributed to P D Boyer.
In the August 10, 2001 issue of Cell, Menz et al. report the crystal structure of a novel inhibited form of the bovine mitochondrial F(1)-ATPase at 2 A resolution, with all three catalytic sites occupied by nucleotide, one of which binds ADP in preference to ATP.
The suggestions from the author's group over the past 25 years for how steps in catalysis by ATP synthase occur are reviewed. Whether rapid ATP hydrolysis requires the binding of ATP to a second site (bi-site activation) or to a second and third site (tri-site activation) is considered. Present evidence is regarded as strongly favoring bi-site activation. Presence of nucleotides at three sites during rapid ATP hydrolysis can be largely accounted for by the retention of ADP formed and/or by the rebinding of ADP formed. Menz, Leslie and Walker ((2001) FEBS Lett., 494, 11-14) recently attained an X-ray structure of a partially closed enzyme form that binds ADP better than ATP. This accomplishment and other considerations form the base for a revised reaction sequence. Three types of catalytic sites are suggested, similar to those proposed before the X-ray data became available. During net ATP synthesis a partially closed site readily binds ADP and Pi but not ATP. At a closed site, tightly bound ADP and Pi are reversibly converted to tightly bound ATP. ATP is released from a partially closed site that can readily bind ATP or ADP. ATP hydrolysis when protonmotive force is low or lacking occurs simply by reversal of all steps with the opposite rotation of the gamma subunit. Each type of site can exist in various conformations or forms as they are interconverted during a 120 degrees rotation. The conformational changes with the ATP synthase, including the vital change when bound ADP and Pi are converted to bound ATP, are correlated with those that occur in enzyme catalysis in general, as illustrated by recent studies of Rose with fumarase. The betaE structure of Walker's group is regarded as an unlikely, or only quite transient, intermediate. Other X-ray structures are regarded as closely resembling but not identical with certain forms participating in catalysis. Correlation of the suggested reaction scheme with other present information is considered.
A suggested minimal scheme for substrate binding by and interconversion of three forms of the catalytic sites of the ATP synthase is presented. Each binding change, that drives simultaneous interchange of the three catalytic site forms, requires a 120 degrees rotation of the gamma with respect to the beta subunits. The binding of substrate(s) at two catalytic sites is regarded as sufficing for near maximal catalytic rates to be attained. Although three sites do not need to be filled for rapid catalysis, during rapid bisite catalysis some enzyme may be transiently present with three sites filled. Forms with preferential binding for ADP and P(i) or for ATP are considered to arise from the transition state and participate in other steps of the catalysis. Intermediate forms and steps that may be involved are evaluated. Experimental evidence for energy-dependent steps and for control of coupling to proton translocation and transition state forms are reviewed. Impact of relevant past data on present understanding of catalytic events is considered. In synthesis a key step is suggested in which proton translocation begins to deform an open site so as to increase the affinity for ADP and P(i), that then bind and pass through the transition state, and yield tightly bound ATP in one binding change. ADP binding appears to be a key parameter controlling rotation during synthesis. In hydrolysis ATP binding to a loose site likely precedes any proton translocation, with proton movement occurring as the tight site form develops. Aspects needing further study are noted. Characteristics of the related MgADP inhibition of the F(1) ATPases that have undermined many observations are summarized, and relations of three-site filling to catalysis are assessed.
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This paper gives an overview of a lecture scheduled for the opening of the 10th European Bioenergetics Congress. In this lecture I plan to first reflect on the accomplishments of some of the individuals who were involved in research on the ATP synthase during the past 50 years. Then I will give a brief view of the present information about rotational catalysis by the ATP synthase. This will be followed by a discussion of some results from my laboratory that call for additional experimentation. Finally I will direct attention to other questions about the ATP synthase that should be addressed in future studies.
Experiments are reported on the uni-site catalysis and the transition from uni-site to multi-site catalysis with bovine heart mitochondrial F1-ATPase. The very slow uni-site ATP hydrolysis is shown to occur without tightly bound nucleotides present and with or without Pi in the buffer. Measurements of the transition to higher rates and the amount of bound ATP committed to hydrolysis as the ATP concentration is increased at different fixed enzyme concentrations give evidence that the filling of a second site can initiate near maximal turnover rates. They provide rate constant information, and show that an apparent Km for a second site of about 2 microM and Vmax of 10 s-1, as suggested by others, is not operative. Careful initial velocity measurements also eliminate other suggested Km values and are consistent with bi-site activation to near maximal hydrolysis rates, with a Km of about 130 microM and Vmax of about 700 s-1. However, the results do not eliminate the possibility of additional 'hidden' Km values with similar Vmax:Km ratios. Recent data on competition between TNP-ATP and ATP revealed a third catalytic site for ATP in the millimolar concentration range. This result, and those reported in the present paper, allow the conclusion that the mitochondrial F1-ATPase can attain near maximal activity in bi-site catalysis. Our data also add to the evidence that a recent claim, that the mitochondrial F1-ATPase does not show catalytic site cooperativity, is invalid.
The mechanism by which ATP is synthesized during oxidative and photophosphorylation has been elucidated by oxygen exchange and other studies: a novel form of catalysis termed rotary catalysis-is involved.
An X-ray structure of the F1 portion of the mitochondrial ATP synthase shows asymmetry and differences in nucleotide binding of the catalytic beta subunits that support the binding change mechanism with an internal rotation of the gamma subunit. Other structural and mutational probes of the F1 and F0 portions of the ATP synthase are reviewed, together with kinetic and other evaluations of catalytic site occupancy and behavior during hydrolysis or synthesis of ATP. Subunit function as related to proton translocation and rotational catalysis is considered. Physical demonstrations of the gamma subunit rotation have been achieved. The findings have implications for other enzymatic catalyses.
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Relatively high ATP concentrations show an unexpected lack of inhibition of the hydrolysis of low concentrations of trinitrophenyl ATP (TNP-ATP) by mitochondrial F1-ATPase. In striking contrast low TNP-ATP concentrations markedly inhibit the hydrolysis of much higher ATP concentrations. The three catalytic sites undergoing sequential conformational changes have different conformations at any instant of catalysis, and only two need to be filled for rapid, steady-state ATP hydrolysis. The remaining site has low affinity for ATP (Kd 2 mM) but about 10(4) greater affinity for TNP-ATP (Km and Kd about 0.2 microM). Thus 500 microM ATP does not prevent binding of less than 1 microM TNP-ATP. As the site binding the TNP-ATP undergoes sequential conformational changes the TNP-ATP undergoes sequential conformational changes the TNP-ATP is hydrolyzed and products are released. The results give strong support to the view that all three catalytic sites proceed equivalently in ATP as well as TNP-ATP hydrolysis. The conformation that has the lowest affinity for ATP has over a 10-fold greater affinity for ADP (Kd 150 microM) and may be akin to the conformation to which ADP binds during net ATP synthesis by the ATP synthase. The recognition of these features was made possible by new information obtained from detailed studies of the interactions of Mg2+, TNP-ADP, TNP-ATP, ATP, and noncatalytic sites on initial and steady-state hydrolysis rates.
The ontogeny of the embryonic and fetal lung involves complex interactions between epithelial and mesenchymal primordia which require a specific program of gene regulation and signal transduction. Past studies in our laboratory using congenic mouse strains indicate that one or more genes which map to the H-2 region of chromosome 17 regulate the rate of lung morphogenesis, defined in this context as differentiative heterochrony among strains. Since hormones and growth factors are the messengers of morphogenesis, it was logical to propose that tumor necrosis factor-alpha (TNF-alpha), a well-characterized cytokine whose gene maps to the D-region of the H-2 complex, is a putative mediator of lung morphogenesis. We investigated this proposition using immunochemical methods and a serumless, chemically defined in vitro model system. Our results demonstrate that: (1) TNF-alpha has a specific spatiotemporal localization, in vivo and in vitro; (2) TNF-alpha receptor, in vivo and in vitro, is localized throughout the embryonic lung; (3) TNF-alpha supplementation in vitro of embryonic lung primordia has a marked dose-dependent, stimulatory effect on branching morphogenesis and surfactant-associated protein (SP-A) expression; (4) multiple immunoreactive proteins, including 17, 26, and 68 kDa species, are expressed during development in vivo, and a subset of these are expressed in vitro; and (5) both time- and glucocorticoid-dependent changes occur in the in vivo expression pattern of TNF-alpha immunoreactive proteins after 4 and 7 days in vitro, including the up-regulation of a novel 40 kDa protein. Given that glucocorticoids (CORT) regulate TNF-alpha expression and TNF-alpha's ability to stimulate pulmonary morphodifferentiation and histodifferentiation, we conclude that TNF-alpha is an autocrine/paracrine pulmonary cytokine, probably a component of the lung morphogenesis pathway regulated by CORT.
Previous results have not established whether the attainment of a rapid photophosphorylation rate as ADP concentration is increased in the micromolar range (apparent Km = approximately 30 microM) results from the filling of a second or a third catalytic site. Measurements reported here show that the ATP synthase of chloroplast thylakoids, with 2-4 microM medium ADP present during steady-state photophosphorylation, has one catalytic site filled with tightly bound nucleotides, but other catalytic sites are largely empty. Thus, the rapid increase in the photophosphorylation rate with higher ADP concentrations results from the filling of a second catalytic site. Even with 30 microM added ADP in the dark, the binding of more than one ADP per synthase was not detectable. The sensitivity of the assay was such that the Kd for binding of ADP at a second catalytic site of the de-energized synthase is > 150 microM, considerably above the apparent Km for rapid photophosphorylation. This result can be explained by an increase in the affinity of a second catalytic site for ADP upon energization. Other experiments assessed the effect of ADP binding at a second catalytic site on the equilibrium between bound ATP and ADP and P(i) at the tight catalytic site. When the rate of photophosphorylation is limited by a low ADP concentration, about equal amounts of ATP and ADP are bound at one catalytic site on the synthase. In contrast, when the rate is limited by a low P(i) concentration with 100 microM ADP present, the equilibrium of bound reactants is shifted so that close to one ATP per synthase is present. This is as expected if the binding of ADP at a second catalytic site allows the protonmotive force to promote ATP formation from ADP and P(i) at a tight binding catalytic site. A scheme for the binding change mechanism incorporating these results is presented.
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The mitochondrial F1-ATPase (MF1) is known to be largely or totally inhibited by combination or reaction with one fluorosulfonylbenzoyl inosine (FSBI), quinacrine mustard, or efrapeptin per enzyme. Measurements were made with 18O in attempt to ascertain if the weak catalytic activity remaining after exposure to excess of these reagents was due to retention of some activity by the enzyme modified by these inhibitors. Any such activity could have different characteristics that might be revealed by the distribution of [18O]Pi isotopomers formed from [gamma-18O]ATP. The MF1 inhibited by FSBI showed progressive appearance of two new catalytic pathways as inhibition proceeded. Both pathways appeared to be operative in the enzyme after one beta subunit per enzyme had been modified by FSBI. A high exchange pathway showed no detectable change as ATP concentration was lowered. The lower exchange pathway showed an increase in the amount of exchange with lowering of the ATP concentration, similar to the cooperative behavior observed with the unmodified enzyme. With excess ATP more product was formed by the low exchange pathway, showing that compulsory alternation between two catalytic sites was not retained. The behavior can be explained by the ability of the modified beta subunit to undergo binding changes similar to those occurring in catalysis, with the other two beta subunits catalyzing sluggish hydrolysis by different pathways because of the asymmetry introduced by the modification. Inhibition by quinacrine mustard also resulted in the appearance of two new pathways, somewhat similar to those from FSBI inhibition. In contrast, activity remaining with excess efrapeptin present showed only one pathway like that of the native enzyme. This can be attributed to a low equilibrium concentration of free enzyme and total inhibition of MF1 combined with efrapeptin.
The presence of ATP at non-catalytic sites of the chloroplast F1-ATPase (CF1) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266, 11551-11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF1. Results reported here show that when assayed in the presence of sulfite, CF1, with some non-catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non-catalytic sites is not necessary for catalytic turnover of CF1. However, even though CF1 with empty non-catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non-catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF1 results from the presence of sulfite, with or without nucleotide binding at non-catalytic sites, the Km remains constant, at about 50 microM for MgATP and 400 microM for MgGTP. The results obtained show that the ATPase activity of CF1 is determined by the fraction of the active enzyme. The inactive CF1.ADP.Mg2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non-catalytic sites promotes the ATP-dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non-catalytic sites results from increasing the portion of CF1 in an active state during steady-state ATP hydrolysis.
Previous studies have not provided definitive information about whether ADP or Pi or their complexes with Mg2+ serve as substrates for photophosphorylation and whether free Mg2+ or ADP is required. Results presented show MgADP, MgGDP, or MgUDP are substrates. At variable Mg2+ concentrations, observed velocities are determined by MgADP and not the free ADP concentration. The approximate Km for MgADP with spinach chloroplasts is about 30 microM, for MgGDP 260 microM, and for MgUDP above 5 mM. The apparent Km values for added ADP or Mg2+ are decreased to constant low values near 30 microM as the added Mg2+ or ADP concentrations, respectively, are increased to the millimolar range. With 100 microM added Mg2+, near-maximal velocities can be obtained with excess ADP, but not with excess GDP or UDP. This is explainable by the apparent Km values for MgGDP and MgUDP being well above 100 microM. High phosphorylation rates with excess of either Mg2+ or ADP present show that little or no (less than 2-3 microM) free Mg2+ or ADP is required. In addition, the results show that during rapid photophosphorylation, when one or more catalytic sites are always filled with nucleotide, free ADP does not combine and block the combination of MgADP to catalytic sites that become vacant. This is in contrast to the ability of free ADP to combine tightly with one catalytic site when all catalytic sites are empty. The apparent Km for added ADP above a few micromolar concentration, and with excess Mg2+ present, results from binding of MgADP at a second catalytic site.(ABSTRACT TRUNCATED AT 250 WORDS)
The interactions between ADP, Mg2+, and azide that result in the inhibition of the chloroplast F1 ATPase (CF1) have been explored further. The binding of the inhibitory Mg2+ with low Kd is shown to occur only when tightly bound ADP is present at a catalytic site. Either the tightly bound ADP forms part of the Mg(2+)-binding site or it induces conformational changes creating the high-affinity site for inhibitory Mg2+. Kinetic studies show that CF1 forms two catalytically inactive complexes with Mg2+. The first complex results from Mg2+ binding with a Kd for Mg2+ dissociation of about 10-15 microM, followed by a slow conversion to a complex with a Kd of about 4 microM. The rate-limiting step of the CF1 inactivation by Mg2+ is the initial Mg2+ binding. When medium Mg2+ is chelated with EDTA, the two complexes dissociate with half-times of about 1 and 7 min, respectively. Azide enhances the extent of Mg(2+)-dependent inactivation by increasing the affinity of the enzyme for Mg2+ 3-4 times and prevents the reactivation of both complexes of CF1 with ADP and Mg2+. This results from decreasing the rate of Mg2+ release; neither the rate of Mg2+ binding to CF1 nor the rate of isomerization of the first inactive complex to the more stable form is affected by azide. This suggests that the tight-binding site for the inhibitory azide requires prior binding of both ADP and Mg2+.
The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.