The proton-translocating ATPase of Escherichia coli.

ANNUAL REVIEWS

Annu. Rev. Biophys. Biophys. Chern. 1990. 19:7-41 Copyright © 1990 by Annual Reviews Inc. All rights reserved

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THE PROTON-TRANSLOCATING

Annu. Rev. Biophys. Biophys. Chem. 1990.19:7-41. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 05/11/15. For personal use only.

ATPase OF ESCHERICHIA COLI A. E. Senior Department of Biochemistry, University of Rochester, Rochester, New York 1 4642 KEY WORDS:

bioenergetics, oxidative phosphorylation, membrane transport.

CONTENTS

............ ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

GENERAL FEATURES OF FUNCTION AND STRUCTURE.........................................................

9

PERSPECTIVES AND OVERVIEW............................

Function ........................................................................... . ................... ............... ... . Structure .......................................................................... . ....................................... .

ROLE OF Fo-SUBUNITS IN PROTON TRANSLOCATION AND F I BINDING ................................

All Three Subunits Are Required for Functional Fo................. . . . . . . ..... . . . . . . . . . . .... . . . . . . . . . Fo Subunits: Structural Models and Involvement in H+ Translocation ...................... Mechanism of H+ Translocation ............ . ......................... . ......... ............................. Role of Fo Subunits in FI Binding ........................................ . ................. ................... .

9 10

11 11 12

15

15

ROLE OF FI SUBUNITS IN NUCLEOTIDE BINDING, CATALYSIS, REGULATION, AND BINDING

OF FI TOFo...................................................................................................

All Five FI Subunits Are Required for A TP-Driven H+ Translocation and ATP Synthesis................................................................................................ Nucleotide-Binding Sites in FI and in Isolated rL and f3 Subunits................................ Structure and Role of Each FI Subunit ............................. ....................................... .

. Reaction Chemistry and "Essential" Residues .............. ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Unisite Kinetics of ATP Synthesis/Hydrolysis: A cceleration to Multisite Activity .... Thermodynamic Aspects of Unisite ATP Hydrolysis and Synthesis........................... Mutational Analysis of Catalysis .............................................................................. Number of Catalytic SUes ......... ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . .. . . . . . . . . . . .

MECHANISM OF CATALYSIS..

.

.. .. .............................................. ....................

PHYSICAL BASIS OF ENERGY COUPLING .. . ..

GENETICS, MUTAGENESIS, EXPRESSION, AND ASSEMBLy.....................................................

16

16 16 18 22 22

24

26

27

29

30 31

The unc Operon........................................................................................................ Methods Used To Obtain Mutants............................................................................ Expression of FIFo Genes ....... ... ................ ......... .................................................... Assembly of FIFo In Vivo .......... ................................................................ .............

31 32 33 34

SUMMARy......................................................................................................................

35

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0883-9 182/90/06 1 0-0007$02.00

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PERSPECTIVES AND OVERVIEW

The proton-translocating adenosine triphosphatase (ATPase) of Escher ichia coli is a plasma membrane enzyme with two physiological roles. Under conditions where respiration can generate a proton gradient (A.uH + ) of sufficient force across the plasma membrane, the enzyme catalyzes A.uH+ -driven ATP synthesis (oxidative phosphorylation), and it is some times referred to as "ATP synthase" for this reason. Under 10w-A.uH+ conditions the enzyme hydrolyzes cytoplasmic ATP and pumps protons into the periplasmic space, thus increasing A.uH + . It is a member of the F 1 F 0 class of ion transport ATPase. The F 1 F 0 class of ATPase is widely distributed, occurring in bacteria, mitochondria, and chloroplasts. All members consist of a membrane-embedded, transmembrane H + -con ducting F 0 sector, linked by a stalk to a membrane-extrinsic F I sector, which contains catalytic sites for ATP hydrolysis and synthesis. Although varying in oligomeric complexity ( 1 25, 240), the enzymes show sufficient overall structural similarity and amino acid sequence homology, par ticularly between subunits most concerned with H+ translocation and ATP synthesis and hydrolysis, that we can be sure that the mechanism of action is the same in all organisms. A salient feature that has proved invaluable in structural and functional studies is that the F 1 and F 0 sectors can be separated, purified, and studied separately. Purified F 1 is water soluble and catalyzes ATPase. Purified F 0 is membranous and carries out passive H+ conduction. Studies of E. coli F 1 F o-ATPase have progressed well in recent years. There are eight subunits in the E. coli enzyme (as compared with ca. 14 in mitochondrial F 1 F 0), and they can be completely dissociated and re associated, with excellent recovery of activity (56, 203). Extensive genetic and molecular biological studies have yielded primary sequences and pre dicted secondary structures of each subunit (246). The eight structural genes are arranged in the unc operon, which may readily be overexpressed from plasmid vectors. Structural advances have come from application of cryoelectron microscopy (86-88), although no high-resolution X-ray structure is available at present. Mutagenesis studies, begun by Gibson, Cox and colleagues in the 1 970s (48), have expanded in recent years and have been combined with kinetic and thermodynamic analyses of the enzyme mechanism. As a result, understanding of the mechanism of H + transport, catalysis, and the nature of energy coupling has become clearer. There have also heen studies of the regulation of expression of the enzyme subunits ( 1 55) and of enzyme assembly in vivo (34). It seemed appropriate to draw this information together here in a review focused explicitly on the E. coli H+ -translocating F IF o-ATPase. Several pertinent reviews are

E. coli H+ -ATPase

9

cited above. Additional recent reviews on F I F o-A TPases are those of Futai et al (76, 77), Senior (207), and Boyer ( 1 3, 1 4). GENERAL FEATURES OF FUNCTION AND STRUCTURE Function

ATP and ADP are preferred substrates for H+ pumping and oxidative phosphorylation, but GTP, GDP, ITP, and IDP are good, probably physiological substrates (185, 254). MgATP, CaATP, MgADP and CaADP are substrates for H+ pumping or oxidative phosphorylation ( 1 85), but the physiological substrates are the Mg-nucleotides, as indicated by the facts that the growth inhibitor azide has little effect on CaATPase (Ki 5 mM), whereas it is a potent MgATPase inhibitor (Ki 25 ,uM) (230) and that growth-retarding mutatIOns that depress MgATPase while leaving CaATPase relatively unaffected are known ( 1 1 7, 250). Other metals that are known to act as cofactors with ATP are Co and Mn (2 1 7).


SUBSTRATES

=

=

INHIBITORS M any of the antibiotic inhibitors that specifically affect mito chondrial or chloroplast F I F 0 are effective against the E. coli enzyme; these include aurovertin, citreoviridin, efrapeptin and venturicidin ( 1 84, 1 97, 1 98, 250). Oligomycin is a weak inhibitor, and ossamycin is not effective ( 1 84). Aurovertin binds specifically to the /3 subunit or the enzyme (55, 1 97), and the fluorescence of bound aurovertin has proved useful in probing enzyme conformational changes. The binding site is around resi due /3-R398 I ( I 3 7a). Venturicidin binds to the c subunit, as shown by its prevention of labeling by dicyclohexylcarbodiimide (DC CD) ( 1 84). The reagents DeCD and 4-chloro-7-nitrobenzofurazan (Nbf) are in activators (253). DCeD binds specifically at residue c-D61 or /3-E I 92 ( 1 03, 256). Azide inhibits "multisite" (steady-state cooperative) ATPase activity, but does not inhibit "unisite" (single-site) activity ( 1 67, 252). Fluoroaluminate is a newly discovered inhibitor ( 1 44).

TURNOVER RATES Vmax rates of ATP hydrolysis by either FIFo in mem branes or purified F I are approximately 50 S-I at pH 7 . 5 and 20ne (52, 18 5). The ATPase activity of purified F I is concentration dependent owing to dissociation of the c-subunit; e-depleted enzyme has three- to fivefold accelerated hydrolysis (58, 135). Vmax rates for ATP hydrolysis are strongly pH dependent, increasing up to ca. pH 9.3 (210). Purified E. coli F I is cold I {1-R398 indicates a residue arginine-398 of the {1 subunit. This notation is used through out. Mutations are described as follows: c-D6IG indicates residue 61 of the c-subunit mutated to glycine from aspartate.


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labile, owing to dissociation into subunits, although not nearly to the same extent as mitochondrial F , . Glycerol and methanol are used to stabilize the enzyme oligomeric structure during purification ( 1 3 1 ) . Membranous F ,F 0 is quite stable at 0° C and when stored frozen. Rates of ATP-driven H+ pumping are consistent with an expected 3H + jATP stoichiometry (203, 207). Measured rates of passive H + conduction through purified F 0 are up to 30 s- I (203). Measured rates of oxidative phosphorylation in E. coli F IF 0 are low, i.e. ca. 1 0 s- ' (254). Rates of ATP synthesis in bovine F IF 0 are ca. 400 s- ' ( 1 52) when extrapolated to theoretical maximal rates under optimal conditions. Bovine F , has an ATPase turnover approxi mately 5 to 1 0-fold higher than E. coli F " thus, the theoretical Vmax for ATP synthesis by E. coli F ,F 0 might be 40-80 s - ' .

Structure F I purified in water-soluble form after release from membranes (see, e.g., 1 6, 78) was found to have the subunit com position a 3[3 3Y& ( 1 7, 68). Its molecular mass is now known to be 382,000 daltons, with 3517 total residues (246). F , F 0 complex purified after deter gent solubilization (67, 71) was shown to have the subunit composition (X3f33y,6 IG, a,b2c", where n = 1 O± 1 (68), or 1 2- 1 5 (238) or 1 2-18 (235). A recent study (93) of the stoichiometry of c subunit failed to resolve n definitively, although it showed that values of 6 which were previously reported in the literature for F ,F 0 enzymes are probably incorrect. The stoichiometries of subunits other than c are well supported by a range of techniques (94, 1 08, 1 1 3, 202, 220). If one assumes a c, 0 stoichiometry, the molecular mass of F , F 0 is 530,000 daltons, with 4890 total residues. F 0 has been purified separately (74, 1 65, 200) and contains a, b, and c subunits in the same stoichiometry as in the F , F 0 complex. That the enzyme con tains eight total subunits, each required for activity, is confirmed by the fact that eight complementation groups have been identified by muta tional analyses (47-49, 1 07). The eight structural genes lie in an operon called unc; overexpression of this operon yielded enhanced levels of F ,F 0 in membrancs with thc eight subunits in the expected stoichio metry (69). SUBUNIT COMPOSITION

ELECTRON MICROSCOPY Electron microscopy of purified F , or of a3f33Y complex showed that the enzyme was spherical, ca. 9 nm in diameter, with a hexagonal subunit arrangement (3, 87, 1 46). By use of monoclonal antibodies or Fab fragments in conjunction with electron microscopy, i t was shown that a and f3 subunits alternate t o form the hexagon (86, 146). The a and f3 subunits are elongated and interdigitated, with calculated dimensions of ca. 9.0 nm by 3.0 nm; an off-center mass at one end of the

E. coli H+ -ATPase

11

central cavity within the hexagon may correspond to the y subunit (86, 87). Cryoelectron microscopy of purified F ,F 0 revealed a membranous F 0 (6 nm in the transmembrane direction) linked by a stalk (ca. 2 nm wide and 4.S nm long) to F 1 ( 1 1 nm by 9 nm) (88). This work showed that F 1 was well separated from F 0 and was connected to it by a surprisingly narrow stalk. In addition to electron microscopy, procedures such as trypsin digestion (82, 94, 98, 1 83), antibody binding (4S, 8S, 1 40, 1 86), chemical labeling (46, 97, 99, 223), and structural characterization of individual purified subunits (2 26, 227) have proved valuable in probing the structure ofF ,F o. Also, predictions of secondary and tertiary structures of the subunits have been made from amino acid sequences ( lIS, 20S, 206, 246). The combined results of electron microscopy, biochemical techniques, and structure prediction suggest the following model for the enzyme. Within the membrane, the predominantly helical and hydro phobic subunits a and c interact with the hydrophobic and helical N terminal 30-residue segment of subunit b (2 copies). Thus, F 0 consists of ca. 1 3 polypeptides (for alb2clO), containing ca. 2S-30 transmembrane helices. Arrangements of the F 0 subunits and helices have been proposed (33, 104, 203). The stalk most probably consists of the hydrophilic, helical, C-terminal 1 26-residue segments of subunits b, which are thought to project out of the membrane, plus the (j s ubunit from F" which is highly helical and elongated. Thus, the stalk may consist of a bundle of four or five helices (88). F \, projected well into the cytoplasm by the stalk (to facilitate i nteraction with nucleotides?), contains the catalytic core a3fJ 3Y, with a hexagonal structure of alternating rx and f3 subunits and a central Y subunit as discussed above, together with subunit 8, whose location is unclear.


MODEL OF THE ENZYME

ROLE OF Fa SUBUNITS IN PROTON TRANSLOCATION AND F, BINDING

All Three Subunits Are Required for Functional Fa Three lines of evidence establish that all three subunits are required for functional F o. First, mutational analysis demonstrated the existence of three structural genes encoding subunits of F 0 and showed that each subunit is required for activity in vivo (35, 47, 50). DNA sequencing characterized these three genes (uncB, uncE, and uncF, corresponding to subunits a, c, and b, respectively) (246). Second, F 0 was dissociated, and individual subunits were purified and reconstituted in proteoliposomes. All three subunits were required for H+ translocation and normal F 1

12

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binding (20 I ) . Third, the mutation b-G9D prevents assembly of F 0 and disrupts its function ( I I I , 1 88). Function is restored by extragenic sup pressor mutations in the a subunit (P240L or P240S) or c subunit (A62P) (1 33, 1 34).

Subunits: Structural Models and Involvement in Translocation


Fo

H+

a subunit (27 1 residues, 30,300 daltons) shows structural analogy to its counterpart ("ATPase 6") in other species, with strong amino acid homol ogy occurring in two regions (residues 1 90-220 and 244-263) toward the C terminus (4 1 , 234). A good structural model is not yet available for this subunit. Models containing 5, 6, or 7 transmembrane helices have been predicted (27, 33, 94, 205, 246), but experimental evidence is lacking. The model presented in Figure 1 a is that of Cain & Simoni (27). In this model, the regions of strongly homologous amino acid sequence occur in helices 5 and 6, and residues known to be sites of mutation conferring oligomycin resistance in higher organisms (expected to be intramembranous and close to the H+ pathway) occur toward the end of helix 4. Numerous mutations have been identified in the a subunit. Several disrupt the assembly of F 0, as judged by their effects on F I F 0 subunit composition or F I binding (59, 62, 235) . Proton translocation is eliminated in all cases in which assembly is defunct, but these mutations unfortunately tell us little about the normal mechanism. Some missense mutations reduce H+ translocation without having concomitant effects on Fo assembly. From studies of such mutations, it has been concluded that residues a R2 1 O, a-E2 l 9, and a-H245 are critical for H+ conduction (see Figure la and its legend). Other residues (GI 97, E 1 96, D I 1 9, S 1 52, and S206; not shown in Figure la) are thought to affect H+ conduction without being critical (25, 1 04, 1 39, 234; R. Fi l lingame, personal communication). Several interesting points have arisen from these mutagenesis studies. First, Cain & Simoni (26) showed that whereas the single mutations a-E2 1 9H and a H245E are each somewhat detrimental (although they do not eliminate H+ translocation as do a-E2 1 9L and a-H254Y), the double mutant a E219H, a-H245 E was more functional than either single mutant, sug gesting a linked functioning of these two residues. Second, residue a-E I 96, which is strongly conserved in different species, could be mutated without causing serious effects (234). Third, the suggested involvement of R, E, and H residues is strikingly similar to the "proton relay" system currently suggested for lac permease ( 1 3 7). However, it is not yet certain whether activity depends only on residues located within the strongly conserved regions toward the C-terminal end of a subunit or whether other parts of the a subunit are also involved.


I -38

iHHHRAf

H245

E219

"::J N

8 8 202

(a)

f-- L31�F lOR)

(OR)(S)

1--128�V,T

D61-+G,N

f-- A25�T I-- A24�S,O

P64-L

>f-- A21�V (S) ;- A20�P

IN

,

C

�

IN

50

R210

205

80/�

�'lOOP

JJ�

30

OUT

C

OUT

N

(b)

2

IN

(c)

Figure 1 Structural models of F 0 subunits, showing residues affected by missense mutations, which inhibit H+ translocation but do not disrupt the F 0 structure, (a) a subunit. References to studies of inhibitory mutations at the residues shown are as follows: R210 (27, 138); E219 (26,139); H245 (25, 26,235), In species other than E. coli, oligomycin resistance-conferring mutations occur at positions 1 95, 1 96, 199, and 244 (reviewed in 207). (b) c subunit. References for inhibitory mutations are as follows: A2 1 V (103); A25T (65); L31F (38, 1 1 2) (this mutation allows F 0 assembly

only in diploid cells); D6IG, N (100, 1 01); P64L (66). In addition to those that inhibit H+ translocation, the following mutations are shown: (S)A20P, which suppresses the P64L mutation (66); (S)A24D, which suppresses the D61G mutation (Fillingame, personal communication); (DR)A24S, which renders F,F 0 DeeD resistant (Fillingame,personal communication); and (DR)I28V,T, which both render F, F 0 DeeD resistant (102). The mutations at positions 31,61, and 64 also confer DeeD resistance. In species other than E. coli, oligomycin resistance-conferring mutations occur at positions 25, 55, 58, 59, 66, and 67 (reviewed in 207). (c) b subunit. No nondisruptive inhibitory mutations have been characterized in this subunit.

�

� ::r:

+

�

'"C

�

....

(;J


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c subunit (79 residues, 8,300 daltons) forms a hairpinlike structure (Figure Ib). The two transmembrane helices are separated by a polar loop oriented toward the cytoplasm. Evidence for this structure has come from the characteristics of the reaction of residue e-D 6 1 with carbodiimides, notably DeeD ( 1 03); from the DeeD resistance-conferring mutations at positions c-28 and c-24 ( 1 02; Fillingame, personal communication); from labeling of e subunit by the hydrophobic 3'-(trifluoromethyl)-3-(m iodophenyl)diazirine (TID) (97, 223), the hydrophilic l -ethyl-3-[3(dimethylamino)propyJ]carbodiimide (EDC) ( 1 42), or tetranitromethane followed by dithionite reduction (46); from antibody-binding experiments against epitopes in the polar loop (85, 1 40); and from effects of mutations in the polar loop (70, 1 60, 1 62). The structures of e subunits from different species are expected to be very similar, given the considerable sequence homology (103). The reaction of DeeD with e-D61 blocks H+ translocation , as does mutation of e-D61 to N or G (l00, 1 0 1 , 1 03). Thus, this carboxyl side chain, situated in the approximate center of the bilayer, seems to play an important rolc. The requirement for a membrane-embedded carboxyl side chain is further supported by the finding that an intragenic suppressor mutation, e-A24D, restores partial function in the e-D6 1 G mutant (Fil lingame, personal communication). Several other e-subunit mutations that affect H + translocation have been characterized. Some, such as those that introduce a new charge into one of the transmembrane helices (e.g. e G23D and e-G58D), fail to assemble the e subunit ( 1 1 2 , 1 61). Some do allow assembly, and these are noted in Figure Ih. It is notable that all these mutations affect residues that are conceivably close to residue D6 1 . They may impact on H + translocation by altering the environment of the D6 1 carboxyl side chain. An intragenic suppressor, e-A20P, restores function to the e-P64L mutant, possibly through effects on kinking of the two helices (66). b subunit ( 1 56 residues, 1 7,200 daltons) has a hydrophobic N-terminal segment (30 residues) followed by a polar 1 26-residue segment. It is pre dicted to be highly helical (205, 245), with the hydrophobic 30-residue N terminus being membrane embedded and interacting with a and e subunits, and the rest projecting from the membrane on the cytoplasmic side and interacting with F I subunits (Figure Ie). Evidence to confirm this model has come from chemical labeling with TID (97) and a hydrophobic male imide (202); from proteolysis studies (94, 98, 1 83); and from antibody binding studies ( 1 86, 224). Analogous subunits in other species are pre dicted to have similar structure but are not strongly homologous in sequence (40, 243). The only charged residue in the intramembranous part of the protein is


E. coli H+-ATPase

15

b-K23, and the mutation b-K23T did not affect H+ translocation (33). Reaction of residue b-C2 l with N-(7-dimethylamino-4-methyl coumaryl)maleimide (DACM) inhibited H+ translocation by only 40% (202). All the b-subunit mutations found to impair function so far also disrupt assembly, i.e. b-G9D, b-G 1 3 1 D ( 1 1 1 , 1 1 3, 1 88), and deletion of the C-terminal Ll56 (23 1). From the data available it appears that b subunit is not directly involved in H + translocation. Interestingly, although b subunit must be assembled with a and c subunits to obtain H+ trans location function, after assembly the C terminus of b subunit may be removed by proteolysis, leaving fragments of ca. 75 or ca. 1 00 amino acids in the membrane (224). Digested b subunit still supports H + translocation by F 0 ( 1 83), although F I binding is abolished. The proteolytic fragments of b subunit do not reconstitute with a and c subunits (224).

Mechanism of H+ Translocation A "proton relay" system, in which protons cross the membrane by sequen tial interaction with a series of buried side chains, is one attractive sugges tion consistent with the mutational analyses described above. Side chains that are involved would include a-R2 1 O, a�E21 9, a-H245, and c-D 6 1 on current information. Diagrams of speculative proton relays were proposed (33, 207). The relative stoichiometry of a to c is ca. 1 / 1 0 (discussed above). DCCD eliminates function by reaction with just one copy of the c subunit (93), and mutations in the c subunit can be dominant (72). Thus, it seems that one copy of the a subunit must interact with a cooperative oligomer of the c subunit. A rotational model was suggested to account for those features (33, 3 7). One could also postulate the formation, by subunits a and c, of a transmembrane water-filled pore, which, with an appropriate H+ filter, would allow specific H+ translocation. Mutational substitutions might be delcterious because they i nterrupted such a pore. One reason for con sidering this alternative is the recent work of Laubinger & Dimroth (136). These workers obtained an F I Fo preparation from Propionigenium mod estum that showed ATP-dependent Na transport at a rate of ea. 35 S-I and a ratio of ca. 1 . 5 for Na transported per ATP hydrolyzed, which elearly favors an ion transport mechanism of the "H20-filled pore" type over a "proton relay" type. On the other hand, in Vibrio parahaemolyticus, which has ATP-driven Na transport and Na-dependent ATP synthesis, mutational analysis has shown these activities are not referrable to the FjFo-like ATPase that is present in this organism ( 1 96).

Role of Fo

Subunits in FI Binding

The predicted structure of b-subunit makes it a prime candidate for F j binding, and there is good experimental evidence that the polar segment


16

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of the b subunit projects from the membrane and interacts with F , (above). b-f3 cross-links have been noted ( 1 1 , 1 4 1 , 1 43). The extent to which portions of subunits a and c interact with F, is unclear. Electron microscopy suggests that such interactions are unlikely, owing to the large spatial separation of F , and F 0 (88). However, mutations in the polar loop of c subunit and antibodies against the c subunit do impede F, binding (85, 1 40, 1 62), and Schneider and Altendorf saw partial F , binding to pro tcoliposomes reconstituted with e, ae, or be subunits (20 1 ). ROLE OF F I SUBUNITS IN NUCLEOTIDE BINDING, CATALYSIS, REGULATION, AND BINDING OF FI TO Fo

All Five FI Subunits Are Required for ATP-Driven H+ Translocation and ATP Synthesis Mutational analysis established the existence offive structural genes encod ing subunits of F, and showed that each subunit is required for activity in vivo (48, 49, 107). Sequencing of the genes gave the amino acid sequences (246; see also 222). The genes are uncH, uncA, uncG, uncD, and uncC, corresponding to 0, cx, y, f3, and B subunits of F b respectively. A major contribution was the body of work on F I subunits by Heppcl and col leagues (56), who dissociated F " purified and characterized each individual subunit, and then reconstituted F, from purified subunits with excellent recovery of activity. All five subunits were required to support ATP-driven H + pumping or ATP synthesis in vitro after reconstitution of F , and rebinding to F o. The smallest subassembly that showed normal ATPase activity was CX3f33Y, but neither this complex nor CX3f33YO nor CX3f33YB would bind to F 0, both 0 and B being needed to achieve proper binding of cx 3/i 3Y to F 0' Neither 0 nor B nor Y would bind to F 0 individually or in combination. Subassemblies of cx, f3, and Y subunits smaller than CX3f33Y were not reported. Using molecular biological approaches, Klionsky and Simoni cor roborated these findings, showing that expression of a, f3, and y subunits is required in cells for full ATPase activity to occur and that 0 and B subunits are both required for correct binding of F, to F ° ( 1 28).

Nucleotide-Binding Sites in FI and in Isolated IX and f3 Subunits F , binds up to six ATP, ADP, or adenyl-5' -imidodiphosphate (AMP PNP) (mol/mol) (109, 1 85, 250, 254). Three sites are readily exchangeable, potentially catalytic sites, which show strong negative cooperativity and ability to bind adenine, guanine, or inosine nucleotide (185, 250). The

E. coli H+-ATPase

17

other three sites exchange slowly with medium nucleotide even during multiple catalytic turnovers, are specific for adenine nucleotide ( 1 85, 254), and are therefore noncatalytic sites. Pure a subunit binds adenine nucleotide at a single site [KdATP 0. 1 pM (55)] with a low dissociation rate (53, 1 73). It does not bind guanine or inosine nucleotide even at millimolar concentrations ( 1 85, 1 90, 204). The nucleotide affinity, low dissociation rate, and specificity together indi cate that the a subunit nucleotide site corresponds to the noncatalytic sites of F I . The nucleotide specificity of isolated a subunit shows that all or most functional elements of the noncatalytic sites are provided by a sub units. The significance of the strong preference for adenine over guanine or inosine nucleotide is not understood. Nucleotide analogs have been used to characterize the a-subunit nucleo tide site. TNP-ATP and TNP-ADP [the 2 3 ,-O-(2,4,6 trinitrophenyl) derivatives of ATP and ADP, respectively] were found to bind to pure a subunit stoichiometrically with high affinity ( 1 90), but 8-azido-ATP had a Kd ::::: 20 mM (1. G. Wise and A. E. Senior, unpublished data). 2-Azido ATP bound stoichiometrically to a-subunit, yet did not yield a stably labeled derivative on photoactivation (249). The analog pyridoxal 5' diphospho-S'-adenosine (PLP-AMP or AP 2-PL) bound stoichiometrically to pure a subunit, with Kd = 1 50 pM, and covalently labeled residue a K20 1 ( 1 9 1 ) . The same residue was labeled when the similar reagent APr PL was used to inactivate F 1 (229), suggesting the conformation of the rx subunit nucleotide site is similar in both isolated r:t. subunit and F I' Once cov alently bound to r:t. subunit, AP 2-PL and AP3-PL appeared to disrupt subunit-subunit interactions, as deduced from the facts that r:t.. APz-PL complex would not reassociate with f3 and y subunits ( 1 9 1 ) and from the reported effects of APrPL on catalysis and nucleotide binding after its reaction with F 1 ( 1 70). Wise et al used 2-azido-ATP to study the non catalytic sites in F I (25 1 ). The analog did not significantly label the rx subunit, consistent with the fact that it did not label isolated a, but it did label residue Y354 of the f3 subunit. The authors noted that "the possibility that . . . noncatalytic binding sites may be formed at interfaces of a and f3 subunits should not be discounted." A similar but not identical suggestion, that parts of the f3 subunit abut on the noncatalytic (rx) nucleotide sites, was made by Rao et al ( 1 90). The disruptive effects of APz-PL (see above) might be explained on this basis. The function of the noncatalytic sites is unknown. These sites exchange slowly both in growing cells ( 1 48) and in membrane vesicles in vitro during oxidative phosphorylation or ATP-driven H+ pumping (254). Rates of GTP synthesis or GTPase seemed relatively unchanged in vitro when the noncatalytic sites were empty or filled with AMP-PNP or ADP (254).


=

',

'

-


18

SENIOR

Therefore, the sites need not be filled for catalysis to occur, and no regu latory function is yet apparent. Assembly of (X3[33Y oligomer from isolated a, [3, and Y subunits in vitro is nucleoside triphosphate dependent (55, 1 90), but this appears to be due to a requirement for nucleoside triphosphate binding to the [3-subunit (catalytic) nucleotide site ( 1 90). Because KdATP is so low, a subunit should always bind ATP in cells, explaining why F 1 F a and purified Flare found to contain up to 3 mol/mol of endogenous adenine nucleotide in noncatalytic sites ( 1 8, 1 09, 1 48, 253). Isolated [3 subunit binds ATP at a single site with a Kd of ca. 40 flM and a fairly high dissociation rate (95, 1 1 0, 168, 1 90). It also binds ADP, TNP-ATP, TNP-ADP, and 3'-0'-(4-[N-(4-azido-2-ni tropheny1) amino-31 butyryl])-ADP (NAP4-ADP) to the extent of 1 mol mol- (1 1 0, 1 45, 190). It is believed that the nucleotide sites on [3 subunits are the catalytic sites on several grounds: (a) the a-subunit site clearly is not catalytic as detailed above; (b) the accumulated evidence (from different F 1 species and several laboratories) of chemical inactivation, affinity labeling, and mutational analyses suggests that thc [3-subunit nucleotide site is the seat of catalysis (reviewed in 207); (c) pure [3 subunit from Rhodospirillum rubrum has been shown to have catalytic activity that is not due to contaminating F 1ATPase (92). In our laboratory we have found that isolated [3 subunit from E. coli hydrolyzes GTP, ITP, or ATP in an aurovertin-sensitive, azide-insensitive fashion, whereas isolated a subunit does not hydrolyze ATP (M. K. AI-Shawi & A. E. Senior, unpublished data). Rates of ATP hydrolysis varied among different [3-subunit preparations from 5 x 1 0 - 4 to 10- 3 s - 1 at pH 8.0 and 20oe. Summarizing, of the six total nucleotide-binding sites in F 1, three poten tially catalytic sites are on [3 subunits and three noncatalytic sites (whose role is unknown) are on a subunits. The predicted locations of the nucleo tide-binding domains in a and f3 subunits are discussed below.

Structure and Role of Each FJ Subunit a-SUBUNIT a subunit (51 3 residues, 55,300 daltons) is the largest F 1 sub unit. Figure 2 shows its three known functional regions and their approxi mate locations. At the N terminus is a membrane-binding region, which is involved in binding F 1 to F Q. Removal of residues 1 - 1 5 by trypsin treat ment of intact F 1 or removal of residues 1 - 1 9 by chymotrypsin treatment of intact F 1 yields an enzyme lacking the (j subunit and, therefore, unable to bind to Fa (57). Subunit isolation and reassociation experiments demon strated that the defect in (j-binding was caused specifically by removal of the a subunit N-terminal residues (57). Digested enzyme retains ATPase activity (57) and has a full complement of six nucleotide sites (A. E. Senior, unpublished data). The mutation (X-G29D has similar effects in that the

E. coli H+ -ATPase

MEMBRANE-BINDING REGION


NH2

1

1

T

30

CT

(15) (19)

NUCLEOTIDE-BINDING DOMAIN

1t

0I./{3

-- ....,�_r-

Gly29

160

Lys 175

19

SIGNAL TRANSMISSION REGION

..1----- COOH 513

Lys201

Ser373

Ser375

Figure 2 Functional regions of Fl ex subunit. Membrane-binding region: T denotes the trypsin cleavage site at residue 1 5 and CT denotes the chymotrypsin cleavage site at residue 1 9 (57). The Gly29 mutation to Asp impairs the binding of F I to the membrane and renders F I deficient of 0 subunit ( 1 50). Nucleotide-binding dumain: The nucleutide-binding dumain

was predicted by Maggio et al ( 1 49) to encompass residues 1 60-340, approximately. A

hypothetical three-dimensional model of this domain has been proposed (189). The Lysl75

mutation to lIc or Glu impairs nucleotide binding ( 1 92). Lys20 1 is specifically labeled by the nucleotide analog AP2-PL ( 1 9I). In the 1J.-{3 signal transmission region, mutations of Gly351

to Asp, Ser373 to Phe, and Ser375 to Phe, block IJ.-{J intersubunit conformational interaction and attenuate positive catalytic cooperativity between catalytic sites on {3 subunits ( 1 49, 252, 253). The mutation ofSer347 to Phe strongly impairs catalysis but is not fully characterized

( 1 49, 253).

mutant F 1 is 6 deficient and binds weakly to F 0 ( 1 50). The N-terminal segment of a subunit probably therefore binds directly to b subunit. Label ing experiments suggest that it may also be close to F 0 (2). It was proposed that the nucleotide-binding domain of a subunit is formed by residues 1 60-340 approximately ( 1 49) from prediction of secondary structure and consideration of sequence homology with other nucleotide binding proteins. In support of this proposal, mutations a-K 1 75I and a K 1 75E have marked effects on nucleotide binding to a subunit ( 1 92), and the nucleotide analogs AP2-PL and APrPL label residue a-K201 (see above). Early studies of a-subunit mutants (24, 208) showed that four missense mutations in the IX subunit impaired catalysis strongly while allowing normal assembly of F IF o. One (S347F) was shown to impair the oligomeric stability of soluble F I and was not further pursued (149, 253). The other three (G35 I D, S3 73F, and S375F), which did form a stable soluble F I , were studied in detail by Wise and colleagues, who showed that in each

20

SENIOR

an intersubunit a/f3 conformational interaction related to catalysis was abolished (253) and that ATP hydrolysis in the multisite, positively co operative mode was strongly inhibited, whereas ATP hydrolysis in the unisite mode was unaffected (211, 252). Although total nucleotide binding was undiminished, negative binding cooperativity between catalytic sites was attenuated (252) Measurements of 180 exchange characteristics of the mutant F I were consistent with these findings (255), as were measure ments of hydrolysis of an ATP analog ( 1 2 1 ) . These mutations cluster together ( 1 49) in a region of a, which is labeled a/f3 signal transmission regiun in Figure 2. It is our hypothesis that this region of a subunit is involved in transmission, across an alf3 interface, of conformational changes signaling positive catalytic cooperativity and negative binding cooperativity between catalytic sites on f3 subunits. Recently, Soga et al (2 1 S) described a mutation (a-R376C) with properties similar to those of the mutations discussed above and situated very close to them. Two other a-subunit mutations that impair catalysis but not assembly (a-P2S 1 L and a-A285V) were also reported in that paper. It should be noted that the nucleotide-binding domain and the a/f3 signal transmission region may not be mutually exclusive, because the mutations a-G3510, ex-S373F, and ex S375F did somewhat reduce the apparent affinity of ATP binding to isolated a subuni t (1 93). Also, if one aligns the a- and f3-subunit sequences, the region a347-a375 aligns with the C-terminal part of the predicted f3subunit nucleotide-binding domain (see below). Amino acid sequences of ex subunits from widely different species are strongly homologous (24 1), and all F I a subunits probably have essentially the same three-dimensional structure. Also, as noted by Walker et al (24 1 , 246), a and f3 subunits are somewhat homologous t o each other and are expected to have similar shapes. In this regard, it is interesting to ask what " structural details determine the different properties (noncatalytic versus catalytic, adenine specific versus nonspecific) of the ()( and f3 nucleotide binding domains.


.

f3 SUBUNIT f3 subunit (459 residues, 50,200 daltons) contains the catalytic nucleotide-binding domain, predicted from secondary structure con siderations to be formed by residues 1 40-335, approximately (5 1 , 207). As pointed out previously (5, 5 1 , 75, 247), short segments of homologous sequence common to numerous diverse nucleotide-binding proteins [called homology A and B by Walker et al (247)] occur at residues 149-156 and 230-242 of f3 subunit. Evidence supporting this proposcd location for the catalytic nucleotide-binding domain has come from covalent labeling ofF I from several species by nucleotide affinity analogs or inactivating reagents (reviewed in 207; see also 30 and 81). Recent work of this type was the


E. coli H +-ATPase

21

labeling of residue fJ-K155 by APrPL in E. coli F , (229). Garboczi et al (79, 80) generated two peptide fragments of rat liver F I f3 subunit, equivalent to residues 1 34- 1 83 and 1 1 5-end of E. coli f3 subunit, each of which showed nucleotide binding. Approximately twenty missense mutations in f3 subunit have been identified that allow assembly of F IF 0 in vivo such that a structurally normal, catalytically impaired F , can be purified. Those mutations occur between residues 1 37 and 246 of the f3 subunit, i.e. within or very close to the predicted nucleotide-binding domain. Thus, the evi dence that the catalytic nucleotide-binding domain is formed by residues 1 40-335, approximately, appears strong. A mutation conferring auro vertin resistance, fJ-R398H, was recently identified (137a). Since binding of nucleotide and aurovertin to isolated f3 subunit is not mutually exclusive ( l 08, 1 1 0), it is unlikely the nucleotide-binding domain includes residue f3398. f3 subunits are homologous to an unusual degree among different species (241), indicating that the three-dimensional structure and the catalytic mechanism are the same in all species. A hypothetical tertiary structure of the E. coli f3-subunit nucleotide-binding domain was presented by Duncan et al (5 1).

ySUBUNIT I' subunit (286 residues, 31,400 daltons) is a component of the minimal lL3f331' catalytic unit, and this complex shows both unisite and multisite kinetics ( 1 68). The underlying reason for the binding of only one copy of yin lL3f33Y is unknown, and it is not known whether the ensuing structural asymmetry induces functional asymmetry or whether the latter is entirely nucleotide induced. I' subunit may function mainly to stabilize the t1. 3f3 3 hexagon. Experiments involving prolonged trypsin digestion of intact F I yielded a functional assembly lL3{33Y', where 1" is a fragment(s) of I' (2 1 6) . Further investigation has now shown that y is cleaved after residue 201 , with both fragments remaining bound in an lL3{33Y'Y" complex (82). The I' subunit and the 8 subunit bind together sufficiently strongly (Kd, ca 3 nM) that Y could be the main binding site for B in F I (54). Mutations identified so far in the Y subunit have been nonsense mutations, deletions, or frameshifts (49,2106,1 1 6, 159). Also, a temperature-sensitive missense mutation of the initiation codon was identified that reduced expression (158). The isolation of missense mutations in y subunit that do not impair the cxpression, assembly, or oligomeric structure of F I would be most valuable. t5 SUBUNIT

t5 subunit ( 1 77 residues, 1 9,300 daltons) is required for binding of F , to F 0 (15, 226). It is elongated and highly helical (226) and most 2 The uncG428 allele contains the mutation y-WJ05end (G. B. Cox, personal com

munication).

22

SENIOR


probably forms part of the stalk linking F I to F 0 (see above). It shows sequence homology to OSCP subunit of bovine F IF 0 ( 1 72, 244), which is thought to form part of the stalk in the bovine enzyme ( 1 47). Trypsin digestion experiments showed that b subunit (as well as y and b subunits and the N-terminal part of IY. subunit) was protected in F IF 0 as compared to F I (82). No missense mutations in b subunit have been reported. e SUBUNIT e subunit ( 1 38 residues, 1 5,000 daltons) is required for binding of F I to F 0 (225), and is globular (227). As noted above, it binds to y subunit, and it is also sufficiently close to one of the three /3 subunits in F I to become cross-linked to it ( 1 4 1 , 1 43, 22 1 ) in the C-terminal region of the f3 subunit (232). The e subunit inhibits multi site ATPase activity of 1Y.3/33Y and 1Y.3/33yb complexes (58, 1 35, 227). Dunn et al (58) concluded that e subunit inhibits by impairing the rate of product release from the catalytic site. Consistent with this, the presence of e enhanced rates of 180 exchange reactions during ATP hydrolysis (255). However, the inhibiting effect on ATPase appears to be relieved in F I F 0 (227), leaving us unclear as to whether e subunit is a true regulatory subunit for membranous enzyme. M utations which cause low expression of e subunit result in very poor growth of cells (84, 1 27). Klionsky et al ( 1 27) found that cellular ATP concentration fell by 40% in such mutant strains and suggested that one role of e subunit is to stifle ATPase activity of soluble ex 3/3 3r complexes that form in the cytoplasm during F IF 0 assembly. A missense mutation, e-G48D, impairs F IF 0 activity and enhances dissociation of e subunit from soluble F I (36). This mutation was suppressed by the second-site mutations e-P47S and e-P47T. Cox et al (36) interpret the results as resulting from disruption by the mutation and then repair by the suppressors of a f3 turn in the e protein chain, which is required for subunit interaction. Kuki et a1 ( 1 32) reported that only residues 1-78 of e subunit are required to support functional binding of F I to F 0 and that residues 80-90 confer the inhibitory properties of e subunit. Residues 9 1 - 1 38 must therefore play an important but as yet unknown role, because one would expect the e subunit to have become truncated before now were this not detrimental. The overall sequence homology between e, b, and y subunits and their counter parts in other species is low (24 1 ) . M ECHANISM O F CATALYSIS

Reaction Chemistry and "Essential" Residues No work on reaction chemistry has been performed on E. coli F b but we may presume that it will resemble that of other F I -ATPases. Data on mitochondrial and thermophilic bacterial F I show that hydrolysis of ATP


E. coli H+-ATPase

23

proceeds by inversion of configuration of the oxygen atoms about the phosphorus atom and suggest that a direct in-line displacement reaction involving a pentacovalent intermediate occurs to yield ADP and Pi' No covalent phosphorylated intermediate is involved, and ADP-O is the phos phate acceptor in ATP synthesis ( 1 4, 213, 24S). Whether there are particular amino acid side chains that play a direct catalytic role, e.g. by protonation-deprotonation, is unknown. Table 1 shows a list of residues that might be suggested to be "essential" on the grounds that they are sites of specific covalent labeling by inactivating reagents. Also shown in Table I are mutational substitutions of these residues and the effects of the mutations on ATPase activity. The results show that none of the residues is critical (with the arguable exception of f3-E I SI). Rather, inactivation of the enzyme by the chemical reagents may be due to introduction of extra matter or perturbation of structure such Table 1 Effects of mutational substitutions of residues in the F 1 f3 subunit that are covalently labeled by chemical modification

Vm•x (ATPase)d

(% of normal)

Residue labeledb

MutationC

FSBA' 2-N,-ATP

Y354

Y354F

III

Nbf' 8-NrATP

Y297

Y297F

76

Nbf' APrPL FDNP-ADP'

KI55

KI55E

25

DeeD'

EI81

El81Q

DeeD

EI92

El92Q

17

F S B I' 2-NrATP Bz-ATPc

Y331

Y331F

26

R281

R281A

45

Reagent'

POc a

------ ---- --

l.l

References for chemical modifications are as follows: FSBA. p-ftuorosulfonylbenzoyl-5' -adenosine

(22); 2-N,-ATP, 2-azido-ATP (251); Nbf, 7-chloro-4-nitrobenzofurazan (8, 9, 228); 8-N]-ATP, 8-azido ATP (96); AP,-PL, adenosine triphosphopyridoxal (229); FDNP-ADP, 3' -O-(5-ftuoro-2,4-dinitrophenyl)

ADP ether (30); DCCD, dieydohexylcarbodiimide (256, 257); FSBI, p-ftuorosulfonylbenzoyl-5'-inosine (23); BzATP, 3'-O-(4-benzoyl)benzoyl-ATP (I); PG, phenylglyoxal (233).

b All residue numbers are for E. coli F 1 {J subunit.

C

All the mutational experiments except R281A were done with E. coli; R281A was done with Sac References for mutations and resultant ATPase activities are as follows: Y354F,

charomyces cerevisiae.

Y297F, Kl55E (176); EI8IQ, El92Q (177); Y33lF (249); R28lA (163). dThe datu for Vm,,(ATPase) arc for purified F 1 where known or, otherwise, for membrane ATPase.

When several mutational substitutions quoted.

of the same

residue were made, the highest activity obtained is

"The chemical modification experiments were performed on mitochondrial or chloroplast Fl' The residues are identical in the E. coli F I {J subunit.


24

SENIOR

that the catalytic nucleotide-binding domain is incapacitated. Whereas one cannot see into the future and say there will not be any essential catalytic residues found, it is interesting to speculate that they may be few or none. The reaction ATP + H 20 ¢ ADP + Pi at the catalytic site is readily reversible, as described below. High-affinity binding, yielding exact stereo chemical orientation, constraint, and polarization of substrates and pro ducts within the catalytic site, may facilitate the reaction. One might envisage that a relatively large number of interactions between amino acid side chains and bound substrate(s) would be needed to achieve this and hence to present an optimum active site surface for binding and catalysis, as discussed, for example, by Fersht (60) for tyrosyl transfer RN A (tRNA) synthetase and Benkovic et al ( 1 2) for dihydrofolate reductase. Prominent among such interactions might be hydrogen bonding and electrostatic bonding. In E I E z-type A TPases, the reaction proceeds via a covalent enzyme phosphate intermediate in which an aspartate residue in the sequence -ICSD*KTGTLT- forms an aspartyl phosphate. The similar sequence -ITST*KTGSIT- occurs in E. coli F I f3 subunit. Noumi et al ( 1 68) made the mutation {3-T285D to test whether this region was involved in catalysis in F l . Despite the fact that the substitution T -> D changed both charge and bulk of the residue, which may have been the reason for the apparent partial disruption of subunit association, the m utation caused only 68% inhibition of ATPase of reconstituted !X 3f3 3') complex and had no effect on ATP bi nding by f3 subunit. The substituting aspartate was not phos phorylated. Therefore, this particular region does not appear to be as important in F I-ATPases as in E I E 2-ATPases. Unisite Kinetics of A TP Synthesis/Hydrolysis: A cceleration to Multisite Activity

Early work to establish kinetic characteristics of F I F o-ATPases was done on mitochondrial and chloroplast enzymes and has been reviewed ( 1 3, 1 4, 42, 207, 2 1 2). The kinetic properties were essentially the same in soluble F I and membranous F IF 0 ( 1 3, 1 4, 1 79). E. coli soluble F I has very similar kinetic properties (7, 52, 252, 255). ATP binds to E. coli FJ at a single catalytic site with high affinity and is hydrolyzed according to the scheme shown i n Figure 3. ATP hydrolysis and its reversal at a single site is referred to as the unisite pathway. The rate constants and equilibrium constants pertaining to the unisite pathway for E. coli F / , together with those for bovine heart mitochondrial F 1 for comparison, are given in Table 2. It is seen that the catalytic step (K2) is freely reversible, consistent with 1 80 exchange assays (255); that Pi release precedes ADP release; and that ADP release is very slow. In the direction

E. coli HT-ATPase F, + ATP

� -

1

F , < ATP

� -2

F , < ADP . Pi

3

Pi

� -3

F , < ADP

4

ADP

� -4

25 F,


Figure 3

Kinetics of ATP hydrolysis and synthesis in F , . This scheme formally represents the unisite pathway, i.e. the pathway occurring when a single catalytic site is occupied by substrate. Rate constants observed under this condition are given in Table 2. Evidence suggests that when two or three of the three potential catalytic sites are filled (called multisite catalysis), catalysis still occurs at unly one sile at any une time but the rate constants are modulated. In the direction of ATP hydrolysis, k+ 2' k+ J, and k + 4 must accelerate; in the direction of ATP synthesis, k_ 3 , k_ , and k _ , must accelerate. K2 appears to remain around 2 unity during this transition to cooperatively modulated catalysis.

of ATP synthesis, k_ 3 is very slow and KdPj is very high, such that synthesis of even bound ATP cannot normally occur from medium ADP plus Pi under unisite conditions. Binding of A TP at a second (and probably third) catalytic site accelerates I the overall rate of ATP hydrolysis to ca. 50 s - in the E. coli enzyme. This enhanced rate, produced by positive catalytic cooperativity, is called the multi site rate. Work on mitochondrial F 1 (43, 44, 1 82) has established that this is due to acceleration of k 2 , k+ 3 , and k 4' Therefore, binding of ATP + + at a distant site (different f3 subunit) elicits conformational change in the

unisite catalytic nucleotide-binding domain, such that the rate constants attain their multi site levels. Data on mitochondrial and chloroplast F IF 0 ( 1 3, 14, l S I ) show that net Table 2

Rate and equilibrium constants for unisite hydrolysis and synthesis of ATP by E. coli and bovine mitochondrial soluble F , Constant

k+ 1 (M - ' s- ') k_ I (s- ' )

E. coli P , " 1.8 7.1

KdATP (M)

3.9

k + 2 (s - ') k _ 2 (S- I)

5.6

K2 k + 3 (5- ') k _ 3 (M ' s - ' ) KdP, (M)

k+4 (5 - 1) k_4 (M - ' s- ' ) KdADP (M) ' Data are

4.7

1 .2

1.0

4.3

2.3 3.4

4.9 6 .9

x

X

x

X

X

Bovine mitochondrial F ,u

10'

6.4

10 - 5

7.5

10- 1 0

1 .2

10 - 2

1.2

10 - 2

2.4 0.5

x

10- 3

2.7

10 - 7

3.5

103

7.8

x

10 - 4

x

102

3.6 2.5

10- '

1.5

x

x X

X

106

X

10 - 6

x

10

X

10- 3

x

10

X

103

x

x

X

X

X

10- ' 2 10

10- 5 10-4 10- 7

taken from Reference 7 (E. coli F ,) and Reference 89

chondrial F I)' Values for mitochondrial k_3 and

described in Reference 7.

k_4

(mito

were recalculated as

26

SENIOR

synthesis (oxidative or photo phosphorylation) requires cooperative interactions of two or more catalytic sites. There is only one operative catalytic pathway under normal conditions, and it is very likely that cataly sis occurs at only one catalytic site at any one time ( 1 4). The actual catalytic equilibrium for synthesis of bound ATP from bound ADP' Pi (K2) appears to be largely insensitive to LlIlH + . Net, rapid, multi site ATP synthesis entails the release of bound ATP from the first catalytic site concomitant with binding of both ADP and Pi into a second catalytic site, and evidence shows that both of these effects are somehow coupled to energy input from 2 LlIlH t ( 1 3 , 14, 1 53). In E. coli F ] , k_ 2 must accelerate from 1 0 - s l under unisite conditions (Table 2) to � IO s- 1 to achieve net ATP synthesis rates measured in membrane preparations. KdPi must change from 2.3 x 1 0 3 M under unisite conditions (Table 2) to a value compatible with the cyto plasmic Pi concentration (ca. 7 mM), and the affinity of the first catalytic site for ATP must change from ca . IO - 1 0 M (Table 2) to ca. 3 mM (the approximate ATP concentration in E. coli cytoplasm) to release bound ATP during oxidative phosphorylation.


A TP

Thermodynamic Aspects of Unisite A TP Hydrolysis and Synthesis Figure 4A shows Gibbs free energy diagrams for the unisite pathway of E. coli and bovine mitochondrial F I . Figure 4B shows the differences 80 60

;Z

40 1 0 E � '-' C>

':i;

-II-

0

A

-+I,..... 0..

i£

-5

-II-

-IIn l£

.... l£

IUU

�

-10 "0 E -15 -,

'I

20

6 � -20

a -20 -40

-25

-60

-30

-80 0..

The KDP ATPase of Escherichia coli.

Catalytic sites of Escherichia coli F1-ATPase.

Mg2+-ATPase defective mutant of Escherichia coli and thiamine transport.

Role of the amino terminal region of the epsilon subunit of Escherichia coli H(+)-ATPase (F0F1).

ATPase of Escherichia coli: purification, dissociation, and reconstitution of the active complex from the isolated subunits.

Role of minor subunits in the structural asymmetry of the Escherichia coli F1-ATPase.

Monomaleimidogold labeling of the gamma subunit of the Escherichia coli F1 ATPase examined by cryoelectron microscopy.

Mutations within the uncE gene affecting assembly of the F1F0-ATPase of Escherichia coli.

The functional groups of the Mg-Ca ATPase from Escherichia coli.

Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector.

Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli.

Role of the carboxyl terminal region of H(+)-ATPase (F0F1) a subunit from Escherichia coli.

Potassium-stimulated N,N'-dicyclohexylcarbodiimide-sensitive AtPase activity in the membranes of anaerobically grown Escherichia coli.

Escherichia coli Type III Secretion System 2 ATPase EivC Is Involved in the Motility and Virulence of Avian Pathogenic Escherichia coli.

Complementation in vitro of mutant and wild-type ATPase of Escherichia coli using isolated subunits.

Catalytic sites of Escherichia coli F1-ATPase. Characterization of unisite catalysis at varied pH.

The expression, purification and crystallization of the epsilon subunit of the F1 portion of the ATPase of Escherichia coli.

Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase.

Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.

Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase.

The products of the kdpDE operon are required for expression of the Kdp ATPase of Escherichia coli.

The MinD protein is a membrane ATPase required for the correct placement of the Escherichia coli division site.

Effects of inducing expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase.

Reconstitution of ATPase activity from the isolated alpha, beta, and gamma subunits of the coupling factor, F1, of Escherichia coli.