The distribution of heavy-chain isoforms of myosin in airways smooth muscle from adult and neonate humans.

ATP synthesis by oxidative phosphorylation in Escherichia coli occurs in catalytic sites on the beta-subunits of F1-ATPase. Random mutagenesis of the beta-subunit combined with phenotypic screening is potentially important for studies of the catalytic mechanism. However, when applied to haploid strains, this approach is hampered by a preponderance of mutants in which assembly of F1-ATPase in vivo is defective, precluding enzyme purification. Here we mutagenized plasmids carrying the uncD (beta-subunit) gene with hydroxylamine or N-methyl-N'-nitro-N-nitrosoguanidine and isolated, by phenotypic screening and complementation tests, six plasmids carrying mutant uncD alleles. When the mutant plasmids were used to transform a suitable uncD- strain, assembly of F1-ATPase in vivo occurred in each case. Moreover, in one case (beta Gly-223----Asp) F1-ATPase assembly proceeded although it had previously been reported that this mutation, when present on the chromosome of a haploid strain, prevented assembly of the enzyme in vivo. Therefore, this work demonstrates an improved approach for random mutagenesis of the F1-beta-subunit. Six new mutant uncD alleles were identified: beta Cys-137----Tyr; beta Gly-142----Asp; beta Gly-146----Ser; beta Gly-207----Asp; beta-Gly-223----Asp; and a double mutant beta Pro-403----Ser,Gly-415----Asp which we could not separate. The first five of these lie within or very close to the predicted catalytic nucleotide-binding domain of the beta-subunit. The double mutant lies outside this domain; we speculate that the region around residues beta 403-415 is part of an alpha-beta intersubunit contact surface. Membrane ATPase and ATP-driven proton pumping activities were impaired by all six mutations. Purified F1-ATPase was obtained from each mutant and shown to have impaired specific ATPase activity.     

The defective proton-ATPase of uncD mutants of Escherichia coli. Two mutations which affect the catalytic mechanism

The catalytic characteristics of F1-ATPases from uncD412 and uncD484 mutant strains of Escherichia coli were studied in order to understand how these beta-subunit mutations cause defective catalysis. Both mutant enzymes showed reduced affinity for ATP at the first catalytic site. While uncD412 F1 was similar to normal in other aspects of single site catalysis, uncD484 F1 showed a Keq of bound reactants greatly biased toward bound substrate ATP and an abnormally fast rate of Pi release. Impairment of productive catalytic cooperativity was the major cause of the reduced steady state ("multisite") catalytic rate in both mutant enzymes. Addition of excess ATP to saturate second and/or third catalytic sites did promote ATP hydrolysis and product release at the first catalytic site of uncD412 F1, but the multisite turnover rate was significantly slower than normal. In contrast, with uncD484 F1, addition of excess ATP induced rapid release of ATP from the first catalytic site and so productive catalytic cooperativity was almost completely absent. The results show that both mutations affect properties of the catalytic site and catalytic site cooperativity and further that the relatively more severe uncD484 mutation affects a residue which acts as a determinant of the fate of bound substrate ATP during promotion of catalysis. Taken together with previous studies of uncA mutant F1-ATPases (Wise, J. G., Latchney, L. R., Ferguson, A. M., and Senior, A. E. (1984) Biochemistry 23, 1426-1432) the results indicate that catalytic site cooperativity in F1-ATPases involves concerted beta-alpha-beta intersubunit communication between catalytic sites on the beta-subunits.     

The defective proton-ATPase of uncA mutants of Escherichia coli. Identification by DNA sequencing of residues in the alpha-subunit which are essential for catalysis or normal assembly

A group of mutant uncA alleles, affecting essential residues of the alpha-subunit of Escherichia coli proton-ATPase, have been identified by intragenic complementation mapping, cloning, and DNA sequencing. One of the mutations, uncA450, abolishes normal assembly of F1-ATPase. The amino acid substitution found was Glu-299----Lys, which is predicted to lie in an alpha-helix in alpha-subunit. The reversal of the charge at residue 299 is a likely cause of defective assembly. The uncA462 allele causes impairment of catalysis while allowing normal assembly of membrane-bound F1-ATPase. The amino acid substitution found was Ser-347----Phe. Three mutations which impair catalysis but do not cause structural perturbation of either membrane-bound or solubilized F1ATPase were characterized as follows: uncA401, Ser-373----Phe; uncA447, Gly-351----Asp; uncA453, Ser-375----Phe. We predict here that the nucleotide-binding domain of alpha-subunit is formed by the amino acids in the sequence from residue 160 to approximately residue 340. The mutations which cause impairment of catalysis lie in a short segment between residues 347-375 of alpha-subunit, at the C-terminal end of the predicted nucleotide-binding domain. This segment is suggested to be important for beta-alpha-beta intersubunit conformational interaction involved in positive catalytic cooperativity in F1-ATPase.     

Directed mutations of the strongly conserved lysine 155 in the catalytic nucleotide-binding domain of beta-subunit of F1-ATPase from Escherichia coli

The amino acid sequence -Gly-X-X-X-X-Gly-Lys- occurs in many, diverse, nucleotide-binding proteins, and there is evidence that it forms a flexible loop which interacts with one or other of the phosphate groups of bound nucleotide. This sequence occurs as -Gly-Gly-Ala-Gly-Val-Gly-Lys- in the beta-subunit of the enzyme F1-ATPase, where it is thought to form part of the catalytic nucleotide-binding domain. Mutants of Escherichia coli were generated in which residue beta-lysine 155, at the end of the above sequence, was replaced by glutamine or glutamate. Properties of the soluble purified F1-ATPase from each mutant were studied. The results showed: 1) replacement of lysine 155 by Gln or Glu decreased the steady-state rate of ATP hydrolysis by 80 and 66%, respectively. 2) Characteristics of ATP hydrolysis at a single site were not markedly changed in the mutant enzymes, implying that lysine 155 is not directly involved in bond cleavage during ATP hydrolysis or bond formation during ATP synthesis. 3) The binding affinity for MgATP was weakened considerably in the mutants (Lys much much greater than Gln greater than Glu), whereas the binding affinity for MgADP was affected only mildly (Lys = Gln greater than Glu), suggesting that lysine 155 interacts with the gamma-phosphate of ATP bound at a single high affinity catalytic site. 4) The major determinant of inhibition of steady-state ATPase turnover rate in the mutant enzymes was an attenuation of positive catalytic cooperativity. 5) The data are consistent with the idea that during multisite catalysis residue 155 of beta-subunit undergoes conformational movement which changes substrate and product binding affinities.     

E. coli F1-ATPase: site-directed mutagenesis of the beta-subunit

Residues beta Glu-181 and beta Glu-192 of E. coli F1-ATPase (the DCCD-reactive residues) were mutated to Gln. Purified beta Gln-181 F1 showed 7-fold impairment of 'unisite' Pi formation from ATP and a large decrease in affinity for ATP. Thus the beta-181 carboxyl group in normal F1 significantly contributes to catalytic site properties. Also, positive catalytic site cooperativity was attenuated from 5 X 10(4)- to 548-fold in beta Gln-181 F1. In contrast, purified beta Gln-192 F1 showed only 6-fold reduction in 'multisite' ATPase activity. Residues beta Gly-149 and beta Gly-154 were mutated to Ile singly and in combination. These mutations, affecting residues which are strongly conserved in nucleotide-binding proteins, were chosen to hinder conformational motion in a putative 'flexible loop' in beta-subunit. Impairment of purified F1-ATPase ranged from 5 to 61%, with the double mutant F1 less impaired than either single mutant. F1 preparations containing beta Ile-154 showed 2-fold activation after release from membranes, suggesting association with F0 restrained turnover on F1 in these mutants.     

Kinetic characterization of the unisite catalytic pathway of seven beta-subunit mutant F1-ATPases from Escherichia coli

We have studied the kinetics of "unisite" ATP hydrolysis and synthesis in seven mutant Escherichia coli F1-ATPase enzymes. The seven mutations are distributed over a 105-residue segment of the catalytic nucleotide-binding domain in beta-subunit and are: G142S, K155Q, K155E, E181Q, E192Q, M209I, and R246C. We report forward and reverse rate constants and equilibrium constants in all seven mutant enzymes for the four steps of unisite kinetics, namely (i) ATP binding/release, (ii) ATP hydrolysis/synthesis, (iii) Pi release/binding, and (iv) ADP release/binding. The seven mutant enzymes displayed a wide range of deviations from normal in both rate and equilibrium constants, with no discernible common pattern. Notably, steep reductions in Kd ATP were seen in some cases, the value of Kd Pi was high, and K2 (ATP hydrolysis/synthesis) was relatively unaffected. Significantly, when the data from the seven mutations were combined with previous data from two other E. coli F1-beta-subunit mutations (D242N, D242V), normal E. coli F1, soluble and membranous mitochondrial F1, it was found that linear free energy relationships obtained for both ATP binding/release (log k+1 versus log K1) and ADP binding/release (log k-4 versus log K-4). Two conclusions follow. 1) The seven mutations studied here cause subtle changes in interactions between the catalytic nucleotide-binding domain and substrate ATP or product ADP. 2) The mitochondrial, normal E. coli, and nine total beta-subunit mutant enzymes represent a continuum in which subtle structural differences in the catalytic site resulted in changes in binding energy; therefore insights into the nature of energy coupling during ATP hydrolysis and synthesis by F1-ATPase may be ascertained by detailed studies of this group of enzymes.     

Structure of the nucleotide-binding domain in the beta-subunit of Escherichia coli F1-ATPase

We propose a working model for the tertiary structure of the nucleotide-binding domain of the beta-subunit of E. coli F1-ATPase, derived from secondary structure prediction and from comparison of the amino acid sequence with the sequences of other nucleotide-binding proteins of known three-dimensional structure. The model is consistent with previously published results of specific chemical modification studies and of analyses of mutations in the beta-subunit and its implications for subunit interactions and catalytic mechanism in F1-ATPases are discussed.     

Oxidative phosphorylation in Escherichia coli. Characterization of mutant strains in which F1-ATPase contains abnormal beta-subunits.

To facilitate study of the role of the beta-subunit in the membrane-bound proton-translocating ATPase of Escherichia coli, we identified mutant strains from which an F1-ATPase containing abnormal beta-subunits can be purified. Seventeen strains of E. coli, characterized by genetic complementation tests as carrying mutations in the uncD gene (which codes for the beta-subunit), were studied. The majority of these strains (11) were judged to be not useful, as their membranes lacked ATPase activity, and were either proton-permeable as prepared or remained proton-impermeable after washing with buffer of low ionic strength. A further two strains were of a type not hitherto reported, in that their membranes had ATPase activity, were proton-impermeable as prepared, and were not rendered proton-permeable by washing in buffer of low ionic strength. Presumably in these two strains F1-ATPase is not released in soluble form by this procedure. F1-ATPase of normal molecular size were purified from strains AN1340 (uncD478), AN937 (uncD430), AN938 (uncD431) and AN1543 (uncD484). F1-ATPase from strain AN1340 (uncD478) had 15% of normal specific Mg-dependent ATPase activity and 22% of normal ATP-synthesis activity. The F1-ATPase preparations from strains AN937, AN938 and AN1543 had respectively 1.7%, 1.8% and 0.2% of normal specific Mg-dependent ATPase activity, and each of these preparations had very low ATP-synthesis activity. The yield of F1-ATPase from the four strains described was almost twice that obtained from a normal haploid strain. The kinetics of Ca-dependent ATPase activity were unusual in each of the four F1-ATPase preparations. It is likely that these four mutant uncD F1-ATPase preparations will prove valuable for further experimental study of the F1-ATPase catalytic mechanism.     

Properties of F1-ATPase from the uncD412 mutant of Escherichia coli.

Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.     

Complete kinetic and thermodynamic characterization of the unisite catalytic pathway of Escherichia coli F1-ATPase. Comparison with mitochondrial F1-ATPase and application to the study of mutant enzymes

A complete analysis is presented of the component rate constants of the "unisite" reaction pathway in normal Escherichia coli F1-ATPase. Gibbs free energy profiles of the unisite reaction pathway were constructed for both normal E. coli F1 and bovine-heart mitochondrial F1, and comparison indicated that E. coli F1 is an ancestral form of the mitochondrial enzyme. Similar kinetic and thermodynamic analyses of the unisite reaction pathway were done for mutant beta-Asn-242 and beta-Val-242 E. coli F1-ATPases. Both mutations affected unisite binding and hydrolysis of MgATP but had little effect on release of products or binding of MgADP. It was apparent that a primary effect of the mutations was on the interaction between the catalytic nucleotide-binding domain and the substrate MgATP. The catalytic transition state [F1-ATP]++ was the most destabilized step in the reaction sequence. Measurements of delta delta G[F1.ATP]++ and linear free energy plots for the catalytic step were consistent with the view that, in normal enzyme, residue beta-Asp-242 accepts an H-bond from the transition-state substrate in order to facilitate catalysis. Both mutations impaired positive catalytic cooperativity. This was caused by energetic destabilization of the catalytic transition state and was an indirect effect, not a direct effect on signal transmission per se between catalytic nucleotide-binding domains on beta-subunits. Therefore, impairment of unisite catalysis and of positive catalytic cooperativity appeared to be linked. This may provide a unifying explanation as to why a series of other, widely separated mis-sense mutations within the catalytic nucleotide-binding domain on F1-beta-subunit, which have been reported to affect unisite catalysis, also impair positive catalytic cooperativity. Linear free energy plots for the ATP-binding step of unisite catalysis demonstrated that beta-Asn-242 and beta-Val-242 mutant enzymes did not suffer any gross disruptive change in structure of the catalytic nucleotide-binding domain, reinforcing the view that impairment of catalysis was due to a localized effect. Such analyses confirmed that six other F1-beta-subunit mutants, previously generated and characterized in this laboratory and thought to have inhibitory side-chain substitutions in the catalytic nucleotide-binding domain, are also devoid of gross structural disruption.     

Characterization of Escherichia coli ATP synthase beta-subunit mutations using a chromosomal deletion strain.

(1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene. JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit. Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes. JP17 was shown to be efficacious for studies of beta-subunit mutations. Site-directed mutants were studied directly in JP17. Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17. (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure. The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism. The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1. However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis. (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site. Neither beta R398C or beta R398W impaired catalysis significantly.     

Preparation and properties of amphipathic enzyme-polymer conjugates.

Five uncoupled mutant strains of Escherichia coli carrying mutations in the uncD gene have been studied. In each of these mutant strains the beta-subunit of the F1 portion of the membrane-bound adenosine triphosphatase is abnormal. In one of the mutant strains (carrying the uncD12 allele) in F1-ATPase aggregate was formed which was purified and found to have low ATPase activity. ATPase activity was absent in the other four strains and the abnormal beta-subunits were tightly bound to the membranes. However, membranes from these strains exhibited various proton permeabilities as indicated by NADH-dependent atebrin-fluorescence quenching and bound different amounts of normal F1-ATPase. The amounts of reconstitution of energy-linked reactions after the addition of normal F1-ATPase also varied depending on the mutant allele. It is apparent that considerable phenotypic variations can occur between strains carrying mutations in the same unc gene.     

Directed mutagenesis of the strongly conserved aspartate 242 in the beta-subunit of Escherichia coli proton-ATPase

Oligonucleotide-directed mutagenesis was used to substitute Asn or Val for residue Asp-242 in the beta-subunit of Escherichia coli F1-ATPase. Asp-242 is strongly conserved in beta-subunits of F1-ATPase enzymes, in a region of sequence which shows homology with numerous nucleotide-binding proteins. By analogy with adenylate kinase (Fry, D.C., Kuby, S.A., and Mildvan, A.S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 907-911), beta-Asp-242 of F1-ATPase might participate in catalysis through electrostatic effects on the substrate Mg2+ or through hydrogen bonding to the substrate(s); an acid-base catalytic role is also plausible. The substitutions Asn and Val were chosen to affect the charge, hydrogen-bonding ability, and hydrophobicity of residue beta-Asp-242. Both mutations significantly impaired oxidative phosphorylation rates in vivo and membrane ATPase and ATP-driven proton-pumping activities in vitro. Asn-242 was more detrimental than Val-242. Purified soluble mutant F1-ATPases had normal molecular size and subunit composition, and displayed 7% (beta-Asn-242) and 17% (beta-Val-242) of normal specific Mg-ATPase activity. The relative MgATPase activities of both mutant enzymes showed similar pH dependence to normal. Relative MgATPase and CaATPase activities of normal and mutant enzymes were compared at widely varied pMg and pCa. The mutations had little effect on KM MgATP, but KM CaATP was reduced. The data showed that the carboxyl side-chain of beta-Asp-242 is not involved in catalysis either as a general acid-base catalyst or through direct involvement in any protonation/deprotonation-linked mechanism, nor is it likely to be directly involved in liganding to substrate Mg2+ during the reaction. Specificity constants (kcat/KM) for MgATP and CaATP were reduced in both mutant enzymes, showing that the mutations destabilized interactions between the catalytic nucleotide-binding domain and the transition state.     

Trinitrophenyl-ATP and -ADP bind to a single nucleotide site on isolated beta-subunit of Escherichia coli F1-ATPase. In vitro assembly of F1-subunits requires occupancy of the nucleotide-binding site on beta-subunit by nucleoside triphosphate

The stoichiometry of nucleotide binding to the isolated alpha- and beta-subunits of Escherichia coli F1-ATPase was investigated using two experimental techniques: (a) titration with fluorescent trinitrophenyl (TNP) derivatives of AMP, ADP, and ATP and (b) the centrifuge column procedure using the particular conditions of Khananshvili and Gromet-Elhanan (Khananshvili, D., and Gromet-Elhanan, Z. (1985) FEBS Lett. 178, 10-14). Both procedures showed that alpha-subunit contains one nucleotide-binding site, confirming previous work. TNP-ADP and TNP-ATP bound to a maximal level of 1 mol/mol beta-subunit, consistent with previous equilibrium dialysis studies which showed isolated beta-subunit bound 1 mol of ADP or ATP per mol (Issartel, J. P., and Vignais, P. V. (1984) Biochemistry 23, 6591-6595). However, binding of only approximately 0.1 mol of ATP or ADP per mol of beta-subunit was detected using centrifuge columns. Our results are consistent with the conclusion that each of the alpha- and beta-subunits contains one nucleotide-binding domain. Because the subunit stoichiometry is alpha 3 beta 3 gamma delta epsilon, this can account for the location of the six known nucleotide-binding sites in E. coli F1-ATPase. Studies of in vitro assembly of isolated alpha-, beta-, and gamma- subunits into an active ATPase showed that ATP, GTP, and ITP all supported assembly, with half-maximal reconstitution of ATPase occurring at concentrations of 100-200 microM, whereas ADP, GDP, and IDP did not. Also TNP-ATP supported assembly and TNP-ADP did not. The results demonstrate that (a) the nucleotide-binding site on beta-subunit has to be filled for enzyme assembly to proceed, whereas occupancy of the alpha-subunit nucleotide-binding site is not required, and (b) that enzyme assembly requires nucleoside triphosphate.     

The properties of hybrid F1-ATPase enzymes suggest that a cyclical catalytic mechanism involving three catalytic sites occurs

Maximal rates of ATP hydrolysis catalyzed by F1-ATPase enzymes are known to involve strong positive catalytic site cooperativity. There are three potential catalytic nucleotide-binding sites on F1. Two important and unanswered questions are (i) whether all three potential catalytic sites must interact cooperatively to yield maximal rates of ATP hydrolysis and (ii) whether a cyclical three-site mechanism operates as suggested by several authors. We have studied these two questions here by measuring the ATPase activities of hybrid enzymes containing normal beta-, gamma-, delta-, and epsilon-subunits together with different combinations of mutant and normal alpha-subunits. The mutant alpha-subunits were derived from uncA401, uncA447, and uncA453 mutant E. coli F1-ATPase, in which positive cooperativity between catalytic sites is strongly attenuated by defined mis-sense mutations. Our data show that three normal catalytic sites are required to interact in order to achieve maximal ATPase rates and suggest that a cyclical mechanism does operate. Hybrid enzyme containing one-third mutant alpha-subunit and two-thirds normal alpha-subunits had substantial but submaximal activity, showing that cooperativity between three sites in a noncyclical fashion, or between pairs of sites, can achieve effective catalysis.     

Directed mutagenesis of the beta-subunit of F1-ATPase from Escherichia coli

Oligonucleotide-directed mutagenesis was used to generate six mutant strains of Escherichia coli which had the following specific amino acid substitutions in the beta-subunit of F1-ATPase: (i) Lys-155----Gln; (ii) Lys-155----Glu; (iii) Gly-149----Ile; (iv) Gly-154----Ile; (v) Tyr-297----Phe;(vi) Tyr-354----Phe. The effects of each mutation on growth of cells on succinate plates or limiting (3 mM) glucose and on cell membrane ATPase activity and ATP-driven pH gradient formation were studied. The results showed Lys-155 to be essential for catalysis, as has been predicted previously from sequence homology and structural considerations; however, the results appear to contradict the hypothesis that Lys-155 interacts with one of the substrate phosphate groups because the Lys-155----Glu mutation was less detrimental than Lys-155----Gln. Gly-149 and Gly-154 have been predicted to be involved in essential conformational changes in F1-ATPase by virtue of their position in a putative glycine-rich flexible loop structure. The mutation of Gly-154----Ile caused strong impairment of catalysis, but the Gly-149----Ile mutation produced only moderate impairment. The two tyrosine residues chosen for mutation were residues which have previously received much attention due to their being the sites of reaction of the inactivating chemical modification reagents 4-chloro-7-nitrobenzofurazan (Tyr-297) and p-fluorosulfonylbenzoyl-5'-adenosine (Tyr-354). We found that mutation of Tyr-297----Phe caused only minor impairment of catalysis, and mutation of Tyr-354----Phe produced no impairment. Therefore, a direct role for either of these tyrosine residues in catalysis is unlikely.     

Importance of F1-ATPase residue alpha-Arg-376 for catalytic transition state stabilization

The functional role of essential residue alpha-Arg-376 in the catalytic site of F1-ATPase was studied. The mutants alpha R376C, alpha R376Q, and alpha R376K were constructed, and combined with the mutation beta Y331W, to investigate catalytic site nucleotide-binding parameters, and to assess catalytic transition state formation by measurement of MgADP-fluoroaluminate binding. Each mutation caused large impairment of ATP synthesis and hydrolysis. Despite the apparent proximity of alpha-Arg-376 to bound nucleoside di- and triphosphate in published X-ray structures, the mutations had little effect on MgADP or MgATP binding affinities, particularly at the highest affinity catalytic site, site 1. Both Cys and Gln mutants abolished transition state formation, demonstrating that alpha-Arg-376 is normally involved at this step of catalysis. A model of the F1-ATPase catalytic transition state structure is presented and discussed. The Lys mutant, although severely impaired, supported transition state formation, suggesting that an additional essential role for the alpha-Arg-376 guanidinium group exists, likely in alpha/beta conformational signal transmission required for steady-state catalysis. Parallels between alpha-Arg-376 and GAP/G-protein "arginine finger" residues are evident.     

The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase.

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]     

Directed mutagenesis of the strongly conserved lysine 175 in the proposed nucleotide-binding domain of alpha-subunit from Escherichia coli F1-ATPase

The alpha-subunit of Escherichia coli F1-ATPase contains an adenine-specific noncatalytic nucleotide-binding domain. A recent proposal (Maggio, M. B., Pagan, J., Parsonage, D., Hatch, L., and Senior, A. E. (1987) J. Biol. Chem. 262, 8981-8984) suggested that this domain is formed by residues 160-340, approximately, in alpha-subunit. Within this proposed domain is a sequence Gly-X-X-X-X-Gly-Lys which is conserved in a large and diverse group of nucleotide-binding proteins and is thought to interact with phosphate groups of bound nucleotide. In this work, residue alpha Lys-175, the terminal residue of the above conserved sequence in F1-alpha-subunit, was mutagenized to Ile or Glu. The specific activity of purified mutant F1-ATPase was reduced by 2.5-fold (Ile) or 3-fold (Glu). Apparent binding of ATP to alpha-subunit, as measured by the centrifuge column procedure, was strongly impaired and ATP-induced conformational change in alpha-subunit, as measured by protection against trypsin proteolysis, was nearly abolished in both mutants. The results suggest that residue alpha Lys-175 is located within the nucleotide-binding domain of alpha-subunit, and that this residue is functionally involved in nucleotide binding. The results support previous suggestions that the alpha-subunit nucleotide-binding site is not involved, directly or indirectly, in catalysis.     

Nuclear genes coding the yeast mitochondrial adenosine triphosphatase complex. Primary sequence analysis of ATP2 encoding the F1-ATPase beta-subunit precursor

The yeast nuclear gene ATP2 encodes a F1-ATPase beta-subunit protein of 509 amino acids with a predicted mass of 54,575 daltons. In contrast to the ATPase beta-subunit proteins determined previously from Escherichia coli and various plant sources, the yeast mitochondrial precursor peptide contains a unique cysteine residue within its immediate amino terminus. Expression of an in-frame deletion in ATP2 between residues 28 and 34 to eliminate this single cysteine residue located near the processing site of the matrix protease does not prevent the in vivo delivery of the subunit to mitochondria or its assembly into a functional ATPase complex. Thus, the import F1 beta-subunit into mitochondria does not require a covalent modification of the type utilized for the secretion of the major lipoprotein from E. coli. In addition, analysis of the level of the major F1-ATPase subunits in mitochondria prepared from an atp2- disruption mutant demonstrates that the in vivo import of these catalytic subunits is not dependent on each other. These data and additional studies, therefore, suggest that the determinants for mitochondrial delivery reside within the amino terminus of the individual precursors.