Alan E. Senior

THE CATALYTIC CYCLE OF P-GLYCOPROTEIN

P‐glycoprotein is a plasma‐membrane glycoprotein which confers multidrug‐resistance on cells and displays ATP‐driven drug‐pumping in vitro. It contains two nucleotide‐binding domains, and its structure places it in the ‘ABC transporter’ family. We review recent evidence that both nucleotide‐sites bind and hydrolyse Mg‐ATP. The two catalytic sites interact strongly. A minimal scheme for the MgATP hydrolysis reaction is presented. An alternating catalytic sites scheme is proposed, in which drug transport is coupled to relaxation of a high‐energy catalytic site conformation generated by the hydrolysis step. Other ABC transporters may show similar catalytic features.

Catalytic mechanism of F1-ATPase

The structure of the core catalytic unit of ATP synthase, alpha 3 beta 3 gamma, has been determined by X-ray crystallography, revealing a roughly symmetrical arrangement of alternating alpha and beta subunits around a central cavity in which helical portions of gamma are found. A low-resolution structural model of F0, based on electron spectroscopic imaging, locates subunit a and the two copies of subunit b outside of a subunit c oligomer. The structures of individual subunits epsilon and c (largely) have been solved by NMR spectroscopy, but the oligomeric structure of c is still unknown. The structures of subunits a and delta remain undefined, that of b has not yet been defined but biochemical evidence indicates a credible model. Subunits gamma, epsilon, b, and delta are at the interface between F1 and F0; gamma epsilon complex forms one element of the stalk, interacting with c at the base and alpha and beta at the top. The locations of b and delta are less clear. Elucidation of the structure F0, of the stalk, and of the entire F1F0 remains a challenging goal.

The molecular mechanism of ATP synthesis by F1F0-ATP synthase

ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F1F0-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.

ReviewCatalytic mechanism of F1-ATPase

The structure of the core catalytic unit of ATP synthase, alpha 3 beta 3 gamma, has been determined by X-ray crystallography, revealing a roughly symmetrical arrangement of alternating alpha and beta subunits around a central cavity in which helical portions of gamma are found. A low-resolution structural model of F0, based on electron spectroscopic imaging, locates subunit a and the two copies of subunit b outside of a subunit c oligomer. The structures of individual subunits epsilon and c (largely) have been solved by NMR spectroscopy, but the oligomeric structure of c is still unknown. The structures of subunits a and delta remain undefined, that of b has not yet been defined but biochemical evidence indicates a credible model. Subunits gamma, epsilon, b, and delta are at the interface between F1 and F0; gamma epsilon complex forms one element of the stalk, interacting with c at the base and alpha and beta at the top. The locations of b and delta are less clear. Elucidation of the structure F0, of the stalk, and of the entire F1F0 remains a challenging goal.

The proton-ATPase of bacteria and mitochondria

ConclusionsWe apologize for the fact that this review is longer than we intended; we excuse ourselves with the statement that since almost all the information we have presented was discovered since one of us last reviewed the field in 1979, we have not strayed from the topical. Remarkable advances in understanding of the mechanism of F1-catalyzed ATP hydrolysis and synthesis, of the structure of the proton-ATPase, of the genetics of the enzyme and its assemblyin vivo have been made in the last four years. Understanding the mechanism of proton translocation across the membrane seems now within our reach, but comprehension of the integration of F1 catalysis and proton translocation remains somewhat elusive. We feel that understanding the roles ofuncF protein, OSCP and F6 is important in this regard. We expect that the rapid chemical characterization of mutation and reversion sites by DNA sequencing will become possible within the near future, and that site-specific mutagenesis may soon allow us to introduce amino acid substitutions in specific domains of specific subunits. Such capabilities would greatly expand our horizons, allowing detailed molecular probing of all the components of the proton-ATPase.We are pleased to have had the opportunity to review the field at an opportune time. Being both Senior and wise we shall not be surprised or distressed if our views and interpretations fail to elicit universal concurrence; but we do hope that all readers find them valuable and stimulating.

ATP synthesis driven by proton transport in F1F0-ATP synthase.

Topical questions in ATP synthase research are: (1) how do protons cause subunit rotation and how does rotation generate ATP synthesis from ADP+Pi? (2) How does hydrolysis of ATP generate subunit rotation and how does rotation bring about uphill transport of protons? The finding that ATP synthase is not just an enzyme but rather a unique nanomotor is attracting a diverse group of researchers keen to find answers. Here we review the most recent work on rapidly developing areas within the field and present proposals for enzymatic and mechanoenzymatic mechanisms.

Both P-glycoprotein Nucleotide-binding Sites Are Catalytically Active

The technique of vanadate trapping of nucleotide was used to study catalytic sites of P-glycoprotein (Pgp) in plasma membranes from multidrug-resistant Chinese hamster ovary cells. Vanadate trapping of Mg- or Co-8-azido-nucleotide (1 mol/mol of Pgp) caused complete inhibition of Pgp ATPase activity, with reactivation rates at 37°C of 1.4 × 10-3 s−1 (t1/2 = 8 min) or 3.3 × 10−4 s−1 (t1/2 = 35 min), respectively. UV irradiation of the inhibited Pgp yielded permanent inactivation of ATPase activity and specific photolabeling of Pgp. Mild trypsin digestion showed that the two nucleotide sites were labeled in equal proportion. The results show that both nucleotide sites in Pgp are capable of nucleotide hydrolysis, that vanadate trapping of nucleotide at either site completely prevents hydrolysis at both sites, and that vanadate trapping of nucleotide in the N- or C-terminal nucleotide sites occurs non-selectively. A minimal scheme is presented to explain inhibition by vanadate trapping of nucleotide and to describe the normal catalytic pathway. The inhibited Pgp·Mg-nucleotide·vanadate complex is probably an analog of the catalytic transition state, implying that when one nucleotide site assumes the catalytic transition state conformation the other site cannot do so and suggesting that the two sites may alternate in catalysis.

ATP synthase: what we know about ATP hydrolysis and what we do not know about ATP synthesis.

In ATP synthase, X-ray structures, demonstration of ATP-driven gamma-subunit rotation, and tryptophan fluorescence techniques to determine catalytic site occupancy and nucleotide binding affinities have resulted in pronounced progress in understanding ATP hydrolysis, for which a mechanism is presented here. In contrast, ATP synthesis remains enigmatic. The molecular mechanism by which ADP is bound in presence of a high ATP/ADP concentration ratio is a fundamental unknown; similarly P(i) binding is not understood. Techniques to measure catalytic site occupancy and ligand binding affinity changes during net ATP synthesis are much needed. Relation of these parameters to gamma-rotation is a further goal. A speculative model for ATP synthesis is offered.

Studies on the mitochondrial oligomycin-insensitive atpase: I. An improved method of purification and the behavior of the enzyme in solutions of various depolymerizing agents

Abstract A new method is described for the preparation of beef-heart mitochondrial ATP-ase. The method consistently gives high yields of enzyme with high activity. The enzyme appears to be homogeneous by several criteria. We have used solutions of phenol-acetic acid-urea, urea alone, guanidine hydrochloride and sodium dodecylsulfate to depolymerize the enzyme. Several subunits are revealed by these techniques. We have tentatively proposed that there are four components in the ATPase complex, with molecular weights of about 8,000, 12,500, 25,000, and 55,000. The heaviest component has been isolated in homogeneous form.

Biological membrane structure. III. The lattice structure of membranous cytochrome oxidase

Abstract Membranous cytochrome oxidase forms a regular two-dimensional array, as visualized by negative staining and electron microscopy. The lattice formed by the spots in the micrographs, which are assumed to be the cytochrome oxidase protein complexes, belongs to the two-dimensional rectangular space group pg; the unit cell dimensions are 88 ± 4 × 127 ± 7 A , with two protein complexes per unit cell. By using the unit cell area in conjunction with the chemical composition of the membrane and the molecular weights and volumes of the constituents, it was possible to construct a geometrical model for the membrane which quantitatively accounts for the measurements in hand. The lipids are present as lamellar bilayer, and the protein complexes pass all the way through the bilayer.