Previous articleNext article No AccessBook ReviewWhole World on Fire: Organizations, Knowledge, and Nuclear Weapons Devastation. By Lynn Eden. Ithaca, N.Y.: Cornell University Press, 2004. Pp. xiv+365. $32.50.David CunninghamDavid CunninghamBrandeis University Search for more articles by this author Brandeis UniversityPDFPDF PLUSFull Text Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by American Journal of Sociology Volume 111, Number 3November 2005 Article DOIhttps://doi.org/10.1086/500770 Views: 33Total views on this site Permission to reprint a book review printed in this section may be obtained only from the author.PDF download Crossref reports no articles citing this article.

The mechanism of ATP hydrolysis by the solubilized mitochondrial ATPase (MF1) has been studied under conditions where catalytic turnover occurs at one site, uni-site catalysis (obtained when enzyme is in excess of substrate), or at two sites, bi-site catalysis (obtained when substrate is in excess of enzyme). Pulse-chase experiments support the conclusion that the sites which participate in bi-site catalysis are the same as those which participate in uni-site catalysis. Upon addition of ATP in molar excess to MF1, label that was bound under uni-site conditions dissociates at a rate equal to the rate of bi-site catalysis. Similarly, when medium ATP is removed, label that was bound under bi-site conditions dissociates at a rate equal to the rate of uni-site catalysis. Evidence that a high affinity catalytic site equivalent to the one observed under uni-site conditions participates as an intermediate in bi-site catalysis includes the demonstration of full occupancy of a catalytically competent site during steady-state turnover at nanomolar concentrations of ATP. Improved measurements of the interaction of ADP at a high affinity catalytic site have lead to the revision of several of the rate constants that define uni-site catalysis. The rate constant for unpromoted dissociation of ADP is equal to that for Pi (4 X 10(-3) s-1). The rate of binding ADP at a high affinity chaseable site (Kd = 1 nM) is equal to the rate of binding ATP (4 X 10(6) M-1 s-1). The rate of catalysis obtained when substrate binding at one site promotes product release from an adjacent site (bi-site catalysis) is up to 100,000-fold faster than unpromoted product release (uni-site catalysis).

Pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), an adenine nucleotide affinity analog, was found to bind in a saturable fashion to isolated alpha-subunit from Escherichia coli F1-ATPase with a stoichiometry of one mol/mol and a Kd approximately 150 microM. The binding was shown to be specific by the following criteria: 1) ATP reduced the binding of PLP-AMP by 80%, and 2) PLP-AMP, like ATP, induced a conformational change which increased the mobility of alpha-subunit in nondenaturing polyacrylamide gel electrophoresis and rendered alpha-subunit resistant to mild trypsin proteolysis. A stable adduct was formed between isolated alpha-subunit and [3H] PLP-AMP after reduction with NaBH4. alpha-Subunit labeled to the extent of 0.4-0.7 mol/mol was digested with trypsin and subjected to high pressure liquid chromatography purification, yielding a single labeled peptide. Automated amino acid sequencing showed that residue alpha-Lys-201 was specifically labeled. The results suggest that Lys-201 occupies a position proximate to the phosphate groups of bound ATP in the alpha.ATP complex. PLP-AMP did not support repolymerization of isolated alpha-, beta-, and gamma-subunits, consistent with previous reports that subunit repolymerization in vitro is dependent upon the presence of nucleoside triphosphate. Further, PLP-AMP-labeled alpha-subunit could not be reconstituted with isolated beta- and gamma-subunits in the presence of ATP, showing that occupation of the alpha-subunit nucleotide site by PLP-AMP impairs normal subunit-subunit interaction.

2-Azidoadenine [32P]nucleotide was bound specifically at catalytic or noncatalytic nucleotide binding sites on beef heart mitochondrial F1 ATPase. In both cases, photolysis resulted in nearly exclusive labeling of the beta subunit. The modified enzyme was digested with trypsin, and labeled peptides were purified by reversed-phase high-pressure liquid chromatography. Amino acid sequence analysis of the major 32P-labeled tryptic fragments showed beta-subunit Tyr-368 to be present at noncatalytic sites and beta Tyr-345 to be present at catalytic sites. From the relationship between the degree of inhibition and extent of modification, it is estimated that one-third of the catalytic sites or two-thirds of the noncatalytic sites must be modified to give near-complete inhibition of catalytic activity.

In order to label a noncatalytic site, F1 containing one vacant noncatalytic site was incubated with a large molar excess of 2-azido-(US ,el-TSP)ATP. Unbound ligand was removed on a Sephadex centrifuge column and MgATP added to displace 2-azido-ANP from catalytic sites. Unbound ligand was again removed on a centrifuge column. Alternatively, F1 was first incubated with MgATP to fill all noncatalytic sites, passed through a centrifuge column, and then incubated with a stoichiometric amount of the 2-azido analog. This resulted in the specific loading of analog at a single high-affinity catalytic site under conditions for uni-site catalysis. The complexes were photolyzed and in both cases, the US subunit was modified. However, separation of tryptic peptides by reversed phase HPLC revealed different labeled peptides. The major labeled peptides were isolated and sequenced. Photolabeling of the noncatalytic site resulted in the modification of Tyr 368, whereas the major labeled tryptic peptide obtained for the catalytic site contained Tyr 345. Near complete inhibition of ATPase activity required the covalent incorporation of either one mol of the photoaffinity analog at a catalytic site or two mol at noncatalytic sites per F1.

This chapter discusses binding change mechanism for adenosine triphosphate (ATP) synthesis by oxidative phosphorylation and photophosphorylation. It presents a three-site version of the binding change mechanism for ATP synthesis by oxidative phosphorylation and photophosphorylation. The chapter presents some experiments, the results of which not only clearly demonstrate the existence of the high-affinity site during steady-state catalysis, but the excellent agreement of the data with the calculated plot also provides an independent confirmation of published values for k1and k3. The effect of an ATP-regenerating system on catalytic site occupancy at low ATP concentration is also presented. F1was incubated with [3H] ATP long enough to allow approximately 10 turnovers per F1. Samples were removed to determine the amount of nucleotide bound to F1and the total [3H] ADP in the reaction mixture. Increasing concentrations of pyruvate kinase decrease medium adenosine diphosphate (ADP) significantly but they have little effect on catalytic site occupancy as shown by the amount of tritiated nucleotide retained by F1upon removal of unbound ligand. These results demonstrate that medium ADP is not required for observing the high-affinity site. Another approach to measure the equilibrium constant for ATP synthesis at the catalytic site in the absence of energization is to determine the amount of bound [32P]ATP formed from the phosphorylation of bound ADP by medium 32Pi. The dependency of the size of this ATP fraction on the Piconcentration suggests that under these conditions, the ΚDfor the Pibinding to a catalytic site containing ADP might be in excess of 0.1 M.