Directed Mutagenesis of the Strongly Conserved Aspartate 242 in the ...

Vol. 263, No . 36, Issue of December 25, PP. 19633-19639,1988 Printed in U.S A .

THEJOURNALOF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Directed Mutagenesis of the StronglyConserved Aspartate 242 in the &Subunit of Escherichia coliProton-ATPase* (Received for publication, July 14, 1988)

Marwan K. Al-Shawi, Derek Parsonage, andAlan E. Senior From the Department of Biochemistry, University of Rochester Medical Center, Rochester, New York 14642

contain the catalytic sites on F1 (reviewed by Senior and Wise, 1983; Vignais and Satre, 1984; see also Harris et al., 1985). Walker et al. (1982) pointed out that the amino acid sequences of several nucleotide-binding proteins, including F1@-subunit, are homologous in two particular regions. These regions are knownfrom x-ray crystallographic studies of adenylate kinase, protein synthesis elongation factor EF-Tu, bacterial phosphofructokinase, andc-H-ras-protein p21 to form structural elements within the nucleotide-binding domains. The first region of homology (“HomologyA”) forms a loop between a @-strand andan a-helix and hasthe sequence Gly-X-X-X-X-Gly-Lys. This region has received attention due to its suggested involvement in the binding of nucleotide phosphate groups (e.g. see Fry et al., 1985; Dreusicke and Schulz, 1986; Jurnak, 1985; LaCour et al., 1985) andits possible involvement in ATP hydrolysis and synthesis in F1ATPase (Fryet al., 1986; Duncan et al., 1986; Hsu et al., 1987; Parsonage et al., 1988a; Garbocziet al., 1988). The second region of homology (“HomologyB”) consists of a sequence of hydrophobic residues followed by an aspartate. This sequence forms part or all of the fourth strand of the psheet in the catalytic nucleotide-binding domain of adenylate kinase, phosphofructokinase, and EF-Tu. In the first two of these enzymes, the aspartatelies closeto thephosphate groups and to the Mgof bound MgATP, whereas in EF-Tu it lies close to the guanine ring (Evans et al., 1981; Sachsenheimer and Schulz, 1977; Pai et al., 1977; Fry et al., 1985; Jurnak, 1985; LaCour et al., 1985). Table I shows this conserved sequence as it is found in the above-mentioned enzymes and in a range of other nucleotide-binding proteins. A region of sequence corresponding to Homology B is present in F1-p-subunits from all species for whichamino acid sequences are known (Walker et al., 1985). The sequence of this region in E. coli F1-@is given in Table I, line 1. Predictions of the secondary and tertiary structureof E. coli F1-@-subunit (Duncan et al., 1986) suggest that this sequence forms a pstrand in the nucleotide-binding domain, with the aspartate residue close to thephosphate groups of bound nucleotide. In the E. coli F1-/3-subunitthis aspartate is residue 242. The membrane-bound FIFo-ATPase of Escherichia coli catFry et al. (1985) proposed that in adenylate kinase the alyzes ATP synthesis during oxidative phosphorylation and amino acids directly involved in catalysis are flanked by the ATP-driven protonextrusion from the cell. FIFo-ATPase can Homology B sequence, which was suggested to be important be separated intotwo sectors. An extrinsic sector (F1)contains in binding or protecting the nucleotide from hydrolysis. They the catalytic sites for ATP synthesis or hydrolysis and has postulated (Fry et al., 1986)that theconserved aspartate (Aspthe subunit composition a3psyGt.The Fo sector is membrane 119 of adenylate kinase) may accept a hydrogen bond from a embedded, conducts protons across the membrane, and is water ligand of M$+ in MgATP, or might directly coordinate responsible for binding of F1to themembrane. The p-subunits the M$+ through the carboxylate group to facilitate the migration of M e from @,y-coordinationin MgATP to a,@* This work was supported by National Institutesof Health Grants coordination in MgADP during catalysis. A similar mechaGM25349 and GM29805. The costs of publication of this articlewere nism may be hypothesized for the equivalent aspartate in F1defrayed in part by the payment of page charges. This article must therefore he hereby marked “aduertisement” in accordance with 18 @-subunit.It is also conceivable that Asp-242 could be inU.S.C. Section 1734 solely to indicate this fact. volved directly in general acid-base catalysis at theF1-ATPase

Oligonucleotide-directed mutagenesis was used to substitute Asn or Val for residue Asp-242 in the 8subunit of Escherichia coli F1-ATPase. Asp-242 is strongly conserved in &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),8-Asp-242 of F1-ATPase might participate in catalysis through electrostatic effects on the substrate Mg2+or through hydrogen bonding to the substrate(s); anacid-base catalytic role is also plausible. Thesubstitutions Asn and Val were chosen to affect the charge, hydrogen-bonding ability, and hydrophobicity of residue 8-Asp-242. Both mutationssignificantlyimpairedoxidative phosphorylation rates in vivo and membrane ATPase and ATP-driven proton-pumping activities in vitro. Asn-242 was more detrimental thanVal-242. Purified soluble mutant F1-ATPases had normal molecular size andsubunit composition, and displayed 7% @-Am242)and 17% (8-Val-242) of normal specific MgATPase 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 K M MgATP, but K M CaATP was reduced. The data showed that the carboxyl side-chain of 8-Asp-242 is not involved in catalysis either as a general acid-base catalyst or through direct involvement in any protonation/deprotonation-linkedmechanism, nor isit likely to be directly involved in liganding to substrateMg2+ during thereaction. 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.

19633

19634

Mutagenesis of E. coli Fl-p Asp-242 TABLEI Conservation of Homology B sequence in nucleotide-binding proteins Sequences and alignments were taken directly from the following references: a Walker et al. (1982); * Walker et al. (1985); Chen, C.-J. et al. (1986); Duncan et al. (1986); e Halliday (1984), Yeast ras YP2 alignment was made here; /Pinkham and Platt(1983),the alignment was made here; Higgins et al. (1985); Evans and Downie (1986); Chen, C.-M. et al. (1986), the alignment was made here. Residue Protein

'

nn

5

1

E. coli F1-ATPase p-subunit" Bovine F,-ATPase p-subunit" E. coli Fl-ATPase a-subunitb Adenylate kinase" ATP/ADP translocasg Phosphofructokinase" EF-Tud Yeast ras YP2' E. coli rho proteid Human mdrl protein' Human mdrl protein' E. coli pstB protein' S. typhirnurium hisP protein8 E. coli malK protein8 S. typhimurium oppD proteing R. leguminosarum nodl proteinh E. coli arsA protein' Consensus*

229-247 239-258 268-286 105-123 282-299 88-106 94-113 77-96 255-274 541-560 1186-1205 164-183 164-183 244-263 177-196 154-173 445-464

10

15

20

K F M DE R R D V - I O N I Y R Y

K I I I I I I L

R A A A A G A A

c L R R R R R M G

V A A G A T A A

E L L I L L L L

H V V A A V L I

K R R I M A C N

K D V I I L L O S I T R L N P K I L L L D E A T S A Q P H I L L L Q E A T S A R P E V L L L @ E P C S A E P D V L L F Q E P T S A E P S V F L L O E P L S N R P K L L I A O E P T T A D P O L L I L a E P T T G

* 8 corresponds to a hydrophobic residue. active site, or in stabilization or destabilization of substrates or reaction intermediates. Residue p-242 might also be involved in conformational signal transmission in F1, since the analogous residue in adenylate kinase (Asp-119) was seen to undergo significant conformation-related movement (Sachsenheimer and Schulz, 1977). We have recently developed a procedure for site-directed mutagenesis and expression of the E. coli F1-ATPase p-subunit (Parsonage et al., 1987b), and here we report the generation of the mutants P-Asp-242+Asn and P-Asp-242+Val. The substitutions Asn and Val are expected to lead to changed charge, hydrogen-bonding capability, and hydrophobicity of residue 242 and were designed to allow discrimination between possible mechanisms of action discussed above and hopefully to lead toan understanding of the role of this strongly conserved aspartate residue. The volumes of buried Asp, Asn, or V l! residues in proteins are reported to be 125, 135, and 142 A3, respectively (Chothia, 1975), so the mutations chosen are not expected to greatly alter the bulk of residue p-242. We describe in this paper the effects of the mutations on oxidative phosphorylation i n uiuo, membrane ATPase, and ATP-driven proton-pumping activities i n vitro, and multisite ATPase activities of purified soluble F1-ATPase. A preliminary report of this work waspublished (Al-Shawi et al., 1988). EXPERIMENTAL PROCEDURES E. coli Strains-The strains used or constructed in this work are described in Table 11. Biochemical Techniques-Growth of E. coli cells, cell membrane preparation, purification of soluble F1-ATPase, and assays of ATPase activity were done as previously described (Wise et al., 1981; Duncan and Senior, 1985).Normal (unc') F1-ATPase was purified from strain SWMl. pH dependence of ATPase activity was assayed as described by Senior et al. (1983). MgATPase and CaATPase activities were investigated at pH 8.0, 30 'C, in 0.5-1 ml of 50 mM Tris-S04containing, F1 (2.5-30 pg) with varied concentrations of ATP, Mg, and Ca as described in the tables and figures. After 5 min the reaction was stopped with an equal volume of 10% (w/v) sodium dodecyl sulfate and the Pi liberated was estimated (Taussky and Shorr, 1953). Concentrations of ionic species ofMg, Ca, and ATP were calculated according to Fabiato and Fabiato (1979). Measurement of ATP- or NADH-induced pH gradient formation

across membranes by acridine orange fluorescence quenching was done as described by Perlin et al. (1983). Measurement of growth yield on limiting glucose wasdone as described by Senior et al. (1984) using 3 mM glucose and adding ampicillin (50 pg/ml) to the medium. Plasmid preparations, cell transformations, and DNA hybridization assays were done as described by Parsonage et al. (1987a). Restriction enzyme digestions, agarose gels, and DNA-ligations were carried out as described by Maniatis et al. (1982). DNA sequencing was done by the dideoxynucleotidechain-termination method (Biggin et al., 1983). Oligonucleotide-directedMutagenesis-Oligonucleotide-directed mutagenesis was carried out by a combination of the methods of Klionsky et al. (1986) and Kunkel et al. (1987) as described by Parsonage et al. (1987b). The template for mutagenesis was a gapped heteroduplex form of phage M13mp18 containing the HindIII-KpnI fragment (encoding the umDCgenes) from plasmid pDP31 (described in Parsonage et al., 1987b). The mutagenic oligonucleotides were as 8follows: P-Asp-242+Asn, 5'-GCTGTTCGTAAACAACATCT-3'; Asp-242+Val, 5'-TGTTCGTTGTTAACATrTAT-3'; (mismatches are underlined). The ratio of mutainic primer to template was 50:l. Mutagenized phage, after ligation, was used to transform strain DP6. Possible mutants were identified by plaque hybridization with 32Plabeled mutagenic oligonucleotide and purified. The presence of the mutations was verified directly by DNA sequencing. In the case of the @-Asp-242+Asnmutation, the elimination of a Him11 restriction site in the replicative form of the phage was diagnostic of the presence of the mutation, and the P-As-Val mutation formed a new HpaI site. The HindIII-KpnI fragment from the replicative form of phage containing each mutation was movedinto plasmid pUC118. The final plasmids, containing the umDC genes in the vector pUC118, were designatedpMKS1 (@-Asp-242+Asn)and pMKS2 (P-Asp-242-Val). The plasmids pMKSl and pMKS2 were used to transform strain AN1272 (umD-C-, see Table 11) yielding strains MKS1 and MKS2 carrying the desired mutations in agenetic background isogenic with that of strain DP1, a normal control strain (Parsonage et al., 1987b). Data Analysis-A multiparameter nonlinear regression program (FITB), kindly supplied to us by Dr. Bruce Simon, University of Maryland School of Medicine, was used for analysis of kinetic and binding data. Materials-DNA polymerase Klenow fragment was obtained from Du Pont-New England Nuclear. T4 DNA-ligase and T4 polynucleotide kinase were obtained from New England Biolabs. Restriction endonuclease enzymes were from Bethesda Research Laboratories and New England Biolabs. a-=S-ATP, [y-32P]ATP, and[2-3H]ADP were purchased from Amersham Corp. 20-mer oligonucleotides for use as DNA-sequencing primers and for directed mutagenesis were

Mutagenesis of E. coli FI-PAsp-242

19635

TABLEI1 E . coli strains used in this work Strain

Genotype

Ref.

SWMl AN1272 AN869 DP6

pAN45 (unc+)/unc+,argH, pyrE, entA, reef unc42O:MuB+E+F+H+A+G+D-C-, argH, pyrE, entA, recA unc418::MuB+E+F+H+A+GfDfC-, argH, pyrE, entA, recA A(uncB-C) ilu::TnlO,recA1, endAl, gyrA96, thi, hsdR17, supE44, relA1, A(lac-proAB),[F’, traD36, proAB, lacIqZAM15]

Rao et al. (1988a) Downie et al. (1981) Gibson et al. (1978) Parsonage et al. (1988b)

pDP3l(uncD+C+)/AN1272 DP1 DP2 MKSl MKS2

pUC118(unc-)/AN1272 pMKSl((3-Asn-242)/AN1272 pMKS2((3-Val-242)/ANl272

synthesized in-house on an Applied Biosystems instrument and purified by polyacrylamide electrophoresis before use. RESULTS

Mutagenesis and Expression of the E. coli uncD Gene Encoding the F1-/3-Subunit-The methods used followed essentially those developed previously (Parsonage et al.,1987b) and are described in detail above (see “Experimental Procedures”). The mutant strainsgenerated contained a mutated uncD (psubunit) gene on a plasmid which encodes and expresses uncD and uncC (F1-t-subunit) genes, in a genetic background in which expression of chromosomal uncD and uncC genes is prevented by the presence of phage Mu. We have previously demonstrated that such a strain incorporates approximately normal (haploid) amounts of F1-ATPase into the cell membranes when the normal uncD gene is present on the plasmid, and shows normal growth characteristics, membrane ATPase, and ATP-driven proton-pumping activities (Parsonage et al., 1987b). In order to demonstrate that the mutantplasmids pMKSl (8-Asn-242) and pMKS2 (p-Val-242) each contained only the single desired mutation, the following procedure was carried out. Four additional M13mp18 plaques of each mutant (giving positive signals in the plaque-hybridization assay using labeled mutagenic oligonucleotide and predicted altered restriction digest patterns) were purified. The presence of the correct mutation was confirmed in one of each group of four by DNA sequencing. The HindIII-KpnI fragment from the replicative form of all eight was transferred to vector pUC118. Each plasmid was then used to transform strain AN1272, and the growth characteristics of each transformant were studied. All four additional isolates of the 8-Asn-242 mutation showed exactly the same impaired growth characteristics on succinate platesor in liquid medium containing limiting glucose as those of strain MKSl(described below); and similarly all four additional isolates of the /3-Val-242 mutation were identical in these characteristics to strain MKS2. The reversion frequency to normal growth on succinate plates was 3.6 X for strain MKSl and 5.1 x lo-‘ for strain MKS2. When plasmids pMKSl and pMKS2 were used to transform strain AN869 (uncD+C-, see Table 11) the resultant strains grew well on succinate plates, showing the uncC gene (F1-c-subunit)was expressed normally from the mutated plasmids. Functional Effects of the p-Asn-242 and p-Val-242 Mutations in Cells and Membrane Preparations-Table I11 shows the effects of the mutations on growth on succinate plates, growth in liquid medium containing limiting (3 mM) glucose, and membrane ATPase activity. Table I11 also shows effects on proton-pumping activities in membrane vesicles. The mutations caused impairment of growth on succinate or limiting glucose, showing that oxidative phosphorylation was impaired in vivo (Cox and Downie, 1979). The A s p A s n

Parsonage et al. (1987b) Parsonage et al. (1987b) Constructed in this work Constructed in this work

mutation wasmore detrimental than Asp+Val, andboth mutations allowed slightly higher growth than fully unccontrol (strain DP2, which does not express either p- or tsubunit of F1), showing that oxidative phosphorylation was proceeding at low rates in both cases. Membrane ATPase activities were significantly lower than normal in the mutants, andactivity was sensitive to inhibition by dicyclohexylcarbodiimide in each case (Table 111). NADHinduced pH gradient formation in membrane vesicles, measured as percent quenching of acridine orange fluorescence, was high(Table 111), demonstrating that themembranes were proton-impermeable and that no open “FO-pores”were present. ATP-induced pH gradient formation in membrane vesicles was significantly impaired in each mutant (Table 111). These results demonstrate that in membranes from the mutant strains, F1 was bound normally to Fo, and F1-ATPase turnover rate was markedly reduced. Purification of Soluble Fl-ATPase from 8-Asn-242 and 8Val-242 Mutant Strains-F1 was purified from strains MKSl and MKS2 by the standardprocedure used in this laboratory (see “Experimental Procedures”). In each case the ATPase activity eluted in a volume of buffer expected for normal F1ATPase in the final step of purification (Sephacryl S-300 gel filtration) showing that the molecular size of the purified F1 was similar to normal. Sodium dodecyl sulfate gels of the purified F1 showedthe subunit composition was the same as in normal F1. In nondenaturing gel electrophoresis (Dunn, 1980) themutant F1-ATPases ranasa single band with mobility identical to thatof normal F1. The specific ATPase activities found were: MKSl F1 (8Asn-242) 1.66 pmol/min/mg (i.e. 6.6% of normal); MKS2 F1 (/3-Val-242),4.25 pmol/min/mg (i.e. 17% of normal). These activities correlate well with the membrane ATPase activities noted above. pH Dependence of ATPase Activity of Soluble Mutant FlATPases-ATPase activity was measured in the pH range 6.9-9.7 and compared to theactivity of normal F1(see Fig. 1). It should be noted that at all pH values, maximal rates were attained. The profile of the relative ATPase activity of @-Am242 F1 was identical to that of normal F1. The p-Val-242 F 1 showed lowered relative ATPase activity at pH values >8.5 as compared to normal, possibly due to a tendency to depolymerize intosubunits at high pH as has been seen with another P-subunit mutant F1 (Wise et al., 1983). Magnesium- and Calcium-ATPase Activities-In order to investigate the possible involvement of residue p-242 in coordinating the cation in the substrate MgATP or CaATP by either direct interaction or by indirect interaction through an H-bonded water-ligand, ATPase activity was measured at constant ATP concentration ( 5 mM) but with total Mg or Ca concentration varied. Fig. 24 illustrates that atall concentrations of Mg used, relative MgATPase activity was the same

19636

Mutagenesis of E. coli Fl-p Asp-242 TABLE 111 Effects of mutations on cell growth, membrane ATPase activity, ann'proton-pumping activity

Strain

Genotype

DP1 DP2 MKS1 MKS2

PAsp-242(unc+) 92 uric-

P-Asn-242 8-Val-242

Growth on succinate plates

++++ 8.6 + 18.6 +

Growth on limiting glucose

Membrane ATPase

Acridine orange fluorescence quenching

NADH-induced 92 47

100 0

0.82

0.42 0.44 0.45

92 98

99 100

100

ATP-induced 87 0 12

Membranes were preincubated for 30 min at 23 "C with 50 p~ dicyclohexylcarbodiimide (DCCD) in 5 mM MgClz, 50 mM Tris-C1, pH 8.0, membrane protein concentration = 0.5 mg/ml.

TABLEIV Comparison of multisite catalytic characteristics of normal, 8-Asn-242, ann' P- Val-242 F1-ATPases Normal 0-Asp-242

Mutant 0-Asn-242

120

A

Mutant 0-Val-242

VmaxMgATP (pmol. 3.0 mg-' .1.5 26 min")' 0.25 0.22 0.12 KMMgATP (mM)* ~ J K MMgATP (s-' M-') 0.65 X 10' 0.07 X lo6 0.09 X 10' VmaxCaATP (pmol.mg" . 2.6 21 1.4 min-') 0.07 0.04 0.13 KMCaATP (mM) ~,/KM CaATP (s-' M-') 1.04 X 10' 0.23 X 10' 0.25 X 10'

O

B

0

V, here is the sum of Vmarland Vmluz of Fig. 3. KMhere is K m of Fig. 3 (low affinity K M ) .

2

4

6

0

10

TOTAL Mg (rnM)

200

120 B

2

I

L

150

F

2 0

e

100

87

50

TOTAL Ca (rnM)

FIG. 2. Cation dependence of ATPase activity. The assay 6 7 8 9 10 conditions were as under "Experimental Procedures" with ATP conPH centration equal to 5 mM. For each enzyme the maximal activity FIG. 1. pH dependence of ATPase activity in purified mu- observed was set at 100%.A, 8-Asp-242 F1; 0, 8-Asn-242 F1; 0, 8Val-242 F,. Panel A, relative ATPase activities plotted against total tant F1-ATPase. The assay medium contained 10 mM NaATP, 5 mM MgSO,, and 50 mM Tris adjusted to pH at 30 "C with HzSO~. amount ofMg present. Actual maximal specific activities (100% 8-Asp-242, Enzyme was added to start the reaction. The relative ATPase activity values) in pmoles of ATP hydrolyzed min".mg" were: 20.5; 8-Asn-242, 1.37; P-Val-242, 2.66. Panel B, relative ATPase at pH 8.0was set at 100% for each enzyme. The actual values measured at pH 8.0 in this assay were: unc+ (P-Asp-242), 26.6 pmol activities plotted against total amount of Ca present. Actual maximal specific activities (100% values) in pmoles of ATP hydrolyzed min-'. of ATP hydrolyzed/min/mg; P-Asn-242,1.7; P-Val-242,3.9; A, &Aspmg" were: P-Asp-242, 13.5; P-Asn-242,0.76; 8-Val-242, 1.71. 242; O,P-Asn-242; 0,P-Val-242.

for all three enzymes. The figure shows activation by MgATP and inhibition by high Mg concentrations. By fitting the activation portion of the curves to the Michaelis-Menten equation, it was found that apparent KM values for the substrate MgATP' for 8-Asp-242, 8-Asn-242, and @-Val-242enzymes were 0.3, 0.5, and 0.4 mM, respectively, and the corresponding pMg values for half-maximal activation were around

' MgATP here denotes the total concentration ofMg bound to ATP species, i.e. the sum of MgATP2- + MgHATP-. Similarly, CaATP denotes the sum of CaATPZ- CaHATP-.

+

5.3 in all cases. pMg for half-maximal inhibition was around 3.1 in allcases. Fig.2B illustrates that inhibition of CaATPase activity by high Ca concentrations was the same for all three enzymes with pCa for half-maximal inhibition of approximately 2.1. However, activation of CaATPase activity was different in the mutant enzymes relative to normal enzyme (Fig. 2B), with both mutant enzymes having higher relative activity than normal enzyme at low CaATP concentrations. Thus, the mutant enzymes seemed to have a higher affinity for CaATP than normal enzyme and 8-Asn-242 enzyme appeared slightly better at binding CaATP than 8-Val-242 enzyme.

Mutagenesis of E. coli F1-P Asp-242 Fig. 3 shows ATPase activity as a function of MgATP concentration, obtained when the ratio of Mg/ATP was kept constant at 0.5, thus yielding an optimal and nearly constant pMg of 4.35. The curved nature of the Eadie-Hofstee plots for all the enzymes indicates apparent negative cooperativity of MgATP binding and positive cooperativity of activation -1.2 during multisite catalysis. Similarly, curved Eadie-Hofstee plots were obtained when the CaATP ratio was maintained at 0.5, yielding an optimal and nearly constant pCa of4.1 (data not shown). Kinetic parameters obtained from these experiments are summarized in Table IV. The mutant enzymes had lower Vmaxvalues for both MgATP and CaATP hydrolysis, with the 8-Asn-242 enzyme having the lowest value in all cases. The KMfor MgATP hardly changed for 8Val-242 enzyme, being 0.22 mM, compared with the normal value of0.25mM, whereas, the KM MgATP in 8-Asn-242 enzyme was decreased to 0.12 mM. However, the values of kcat/& indicate that the specificity for MgATP during multisite hydrolysis was lower in the mutant enzymes (Fersht, 1985). Relative specificities for MgATP were 1.0, 0.14, and 0.11 for 8-Asp-242, P-Val-242, and 8-Asn-242 enzymes, respectively. K M values for CaATP (Table IV) were markedly lower in the mutantenzymes as compared to normal enzyme 400 A 300

t \

200

100

0 0

50 . 40

5

10

20

15

25

30

81

0

.

30 . 20

19637

TABLE V Energies of interaction with the transition state during multisite catalysis CaATP us. MgATP MgATP CaATP

F,-ATPase

AAGb" kJ mol"

AGI

AG?

kJ mol"

kJ mol"

(3-Asp-242 0 0 5.4 -2.9 3.8 (3-Asn-242 5.1 -2.7 3.6 (3-Val-242 AAGb is the apparent difference in binding energy for MgATP and CaATP. AAGb is calculated from the values of Table IV. Assuming that the molecular mechanism for MgATP and CaATP hydrolysis has the same rate-limiting step, and ignoring any possible inductive effects, then: AAGb = -RT

ln[(~~t/K~)o~p/(k,/~M)M~TPl

(Fersht, 1985). AG1is the interaction energy with the transition state for multisite MgATP hydrolysis, calculated from the values given in Table IV using the following equation: AGI = -RT l n [ ( k ~ ~ / K ~ ) ~ " ~ " ~ / ( k ~ t / K ~ ) " ~ ~

(Lowe et al., 1985). AG, in this column represents the interaction energy with the transition state for multisite CaATP hydrolysis.

(0.04,0.07, and 0.13 mM for P-Asn-242,8-Val-242,and P-Asp242 enzymes, respectively). kat/&values for CaATP hydrolysiswerelowerfor the mutant enzymes, and the relative specificities were 1.0,0.24, and 0.22 for 8-Asp-242, 8-Val-242, and 8-Asn-242 enzymes, respectively. The results of Table IV can also be given in the thermodynamic terms of Table V, which clearly illustrates the trends observed above (see "Discussion" for further details). It is interesting to note that the specificity of all the enzymes studied here was higher for CaATP than MgATP. Ratios of CaATP specificity/MgATP specificity were 3.3,2.8, and 1.6 for 8-Asn-242, 8-Val-242, and B-Asp-242 enzymes, respectively. It should also be noted that theKMvalues given for normal enzyme in Table IV are lower than have been previously reported (Wise et al., 1983; Kanazawa et al., 1980), the reason being that those authors used the concentration of total nucleotide in their calculations. Dunn et al. (1987) obtained apparently lower VmaxMgATPase values in c-subunitreplete enzyme than those reported here, but this could be due to the relatively high free Mg (2 mM) used in that work, which would be expected from our experiments to have been inhibitory (KI Mg = 0.8 mM).

'

DISCUSSION 10. 0 0.0

0.5 1.5 1.0

2.0

2.5 3.5 3.0

v &rnol.mg-1.rnin-1)

FIG. 3. Eadie-Hofstee plot of multisite MgATPase activities of Fl-ATPases. The assay conditions were the same as in Fig. 2 except that concentration of MgATP was varied from 0.05-10 mM. The ratio of Mg to ATP was kept constant at 0.5 resulting in an optimal and nearly constant pMg of4.35. [MgATP] indicates the total concentration of MgATP species (MgATP2- + MgHATP-). A, (3-Asp-242F1; 0, 8-Asn-242 F1; 0, (3-Val-242 F1. The lines drawn through the data points are computed least squares regression fits to the datausing the following equation:

where Vma.l, K Mare ~ higher affinity components, and Vmarzand K M are lower affinity components.

In the Introduction to this paper, we discussed the arguments which suggest that the strongly conserved residue 8Asp-242 and the group of hydrophobic residues which immediately precede it in the amino acid sequence may form an important structural and possibly functional element in the catalytic nucleotide-binding domain of the 8-subunit of F1ATPase. We further described plausible roles that thisaspartate residue could play in catalysis if it lies close to the phosphate groups and coordinated Mg of bound nucleotide, as structuralconsiderations suggest it may. The mutations 8Asp-242-Asn or Val were chosen to test these ideas. Each of the purified soluble mutant S-ATPase enzymes obtained in this work seemed to be of normal molecular size, subunit composition, and oligomeric stability, as shown by the behavior during gel filtration, sodium dodecyl sulfate-gel electrophoresis, nondenaturing gel electrophoresis, and pH~ dependent ATPase assays described under "Results," with the possible exception that 8-Val-242 enzyme may depolymerize

19638

Mutagenesis of E. coli F,-P Asp-242

to some extent at pH > 8.5. In each case, mutant F1 appeared to bind with normal affinity to membranes and to block proton conduction through Fo. Therefore, we may conclude that neither mutation affected assembly of the enzyme or brought about any gross perturbation of its structure (see also the accompanying paper). Analysis of possible effects of the mutations on secondary structure by the method of Chou and Fasman (1978) showed that Asn at residue 242 would not change the secondary structure, whereas Val would tend to extend the predicted P-strand in this region by at least 1 residue. The results reported in this paper show that both mutations significantly reduce the rates of ATP synthesis and ATP hydrolysis on F1, with the (3-Asn-242 being more detrimental for both activities than the 8-Val-242 mutation. It is clear that a carboxyl side-chain at residue 242 is not absolutely required for catalysis since both mutantenzymes retained low rates of ATP hydrolysis and oxidative phosphorylation. From the facts that P-Asn-242 mutant enzyme displayed low catalytic activity and that its ATPase activity showed the same relative pH dependence between pH values of 6.5 and 9.7, we can rule out the suggestion that residue 242 is involved in general acid-base catalysis. It would also appear that residue 242 is not directly involved in catalysis through any protonation/deprotonation-linkedmechanism. The results with 8Val-242 mutant enzyme also strongly support these conclusions. The mutations had little effect on relative MgATPase activities of the enzymes (Fig. 2), and KM values for MgATP under multisite conditions were similar (Table IV). These facts, taken together with the finding that under “unisite” MgATPase conditions the equilibration of bound substrates with bound products (Keqfor the hydrolytic reaction) was hardly changed by the mutations (see accompanying paper), suggest that theintrinsic catalytic mechanism was not altered. It is therefore unlikely that residue 8-Asp-242 is involved directly or indirectly in liganding substrate M e during the reaction. As acontrasting example, Serpesu et al. (1986) mutated the Ca2+-ligandingAsp-40 of Staphylococcal nuclease to Gly, and found 10-fold increase in KMfor Ca at saturating DNA concentration. All the enzymes had a higher specificity for CaATP than MgATP (Fig. 2, Tables IV and V), and the apparent difference in binding energies of the two substrates (AAGb) were -1.2, -2.9 and -2.7 kJ mol” for normal, P-Asn-242, and 8-Val-242 enzymes, respectively.This higher specificity for CaATP may be attributable to the higher degree of flexibility of Ca over Mg in accepting ligands. Ca can accept up to eight ligands from flexibleangles and with flexible bond lengths, compared to only six ligands at fixed angles and with constrained bond lengths for Mg (Williams, 1976; 1980). The mutant enzymes had lower specificities for MgATP and CaATP than normal enzyme (Table IV). AGI (the interaction energy with the transition state) was destabilized in the mutant enzymes by approximately 5 and 4 kJ mol-’ for MgATPase and CaATPase activity, respectively (Table V). This seems to indicate that a change had occurred in the catalytic nucleotide-binding domains’ interactions with the substrates during the reaction in the mutant enzymes. From the results, it would appear that of the two mutants, 8-Asn242 had the more changed nucleotide-binding domains in the multisite transition state. This point is further investigated in the accompanying paper (Al-Shawi and Senior, 1988). While this work wasin progress a report appeared by Yohda et al. (1988), in which residue Asp-252 of @-subunitof PS3 F1-ATPase (equivalent to P-Asp-242of E. coli F1)was mutated

to Asn. These workers found, in contrast to what we found here, that this Asp+Asn mutation caused complete inactivation of ATPase activity. Wehave also found that the mutations a-Lys-l75+Ile, P-Glu-l81+Gln and D-Glu-192+ Gln in E. coli F1, like P-Asp-242+Asn, yield F1-ATPase with impaired yet readily detectable activities; whereas Yohda and co-workers find that the equivalent mutations in PS3 a- or 8-Fl-subunits all cause complete inactivation of ATPase (Parsonage et al., 1987b, 1988b; Rao et al., 1988b; Ohtsubo et al., 1987; Yohda et al., 1988). These differences may be due to species differences between PS3 andE. coli, or to thefact that assembly of catalytic oligomers from F1-subunits in vitro, as used by Yohda and colleagues to study their mutations, may involve a different pathway from that of FIFo-ATPase assembly in vivo (Cox and Gibson, 1987). Acknowledgment-We thank Professor A. S. Mildvan for valuable discussions. REFERENCES Al-Shawi, M. K., and Senior, A. E. (1988)J. Biol. Chem. 263,1964019648 Al-Shawi, M. K., Parsonage, D., and Senior, A. E. (1988) Biophys. J. 5 3 , 3 3 (abstr.) Biggin, M. D., Gibson, T. J., and Hong, G. F. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,3963-3965 Chen, C-J., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B. (1986) Cell 4 7 , 381-389 Chen, C-M., Misra, T. K., Silver, S., and Rosen, B. P. (1986) J. Biol. Chem. 261,15030-15038 Chothia, C. Y. (1975) Nature 254,304-308 Chou, P. Y., and Fasman, G.D. (1978) Adu. Enzymol. 45,145-148 Cox, G. B., and Downie, J. A. (1979) Methods Enzymol. 5 6 , 106-117 Cox, G. B., and Gibson, F. (1987) Current Topics Bioenerg. 15,162175 Downie, J. A., Cox, G. B., Langman, L., Ash, G., Becker, M., and Gibson, F. (1981) J. Bacteriol. 145,200-210 Dreusicke, D., and Schulz, G. (1986) FEBS Lett. 2 0 8 , 301-304 Duncan, T. M., Parsonage, D., and Senior, A. E. (1986) FEBS Lett. 208,l-6 Duncan, T. M., and Senior, A.E. (1985) J . Biol. Chem. 2 6 0 , 49014907 Dum, S.D. (1980) J. Biol. Chem. 255, 11857-11860 Dunn, S. D., Zadorozny, V. D., Tozer, R. G., and Orr,L. E. (1987) Biochemistry 26,4488-4493 Evans, I. J., and Downie, J. A. (1986) Gene 43,95-101 Evans, P. R., Farrants, G. W . , and Hudson, P. J. (1981) Phil. Trans. R. SOC.Lond. B293,53-62 Fabiato, A., and Fabiato, F. (1979) J. Physiol. (Paris) 76,463-505 Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd ed., W . H. Freeman, New York Fry, D. C., Kuby, S. A., and Mildvan, A. S.(1985) Biochemistry 2 4 , 4680-4694 Fry, D. C., Kuby, S. A., and Mildvan, A. S. (1986) Proc. Natl. Acad. Sci. U. S. A . 83,907-911 Garboczi, D. N., Shenbagamurthi, P., Kirk, W . , Hullihen, J., and Pedersen, P. L. (1988) J. Bwl. Chem. 263,812-816 Gibson, F., Downie, J. A., Cox, G. B., and Radik, J. (1978)J. Bacteriol. 134, 728-736 Halliday, K. R. (1984) J. Cyclic Nucleotide Protein Phosphorylation Res. 9,435-448 Harris, D. A., Boork, J., and Baltscheffsky, M. (1985) Biochemistry 24,3876-3883 Higgins, C. F., Hiles, I. D., Whalley, K., and Jamieson, D. J. (1985) EMBO J. 4,1033-1040 Hsu, S-Y., Noumi, T., Takeyama, M., Maeda, M., Ishibashi, S., and Futai, M. (1987) FEBS Lett. 2 1 8 , 222-226 Jurnak, F. (1985) Science 2 3 0 , 32-36 Kanazawa, H., Moriuchi, Y., Takagi, M., Ishino, Y., and Futai, M. (1980) J. Biochem. (Tokyo) 88, 695-703 Klionsky, D. J., Skalnik, D. G., and Simoni, R. D. (1986) J. Biol. Chem. 261,8096-8099 Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Methods Enzymol. 154,367-382

Mutagenesis of E. coli FI-p Asp-242 LaCour, T. F. M., Nyborg, J., Thirup, S., and Clark, B. F. C. (1985) EMBO J. 4,2385-2388 Lowe, D. M., Fersht, A. R., Wilkinson, A. J., Carter, P., and Winter, G. (1985) Biochemistry 2 4 , 5106-5109 Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Ohtsubo, M., Yoshida, M., Ohtat s., Kagawa, y.,Yohda, M., and Date, T. (1987) Biochem. Biophys. Res. Commun. 1 4 6 , 705-710 Pai, E. F., Sachsenheimer, w., Schirmer, R. H., and Schulz, G. E. (1977) J. Mol. Biol. 1 1 4 , 37-45 Parsonage, D., Duncan, T. M., Wilke-Mounts, s.,Kironde, F. A. s., Hatch, L., and Senior, A. E. (1987a) J. Bwl. Chem. 2 6 2 , 63016307 Parsonage, D., Wilke-Mounts, S., and Senior, A. E. (1987b) J . Bid. Chem. 262,8022-8026 Parsonage, D., Al-Shawi, M.K., and Senior, A. E. (19884 J. Biol. Chem. 263,4740-4744 parsonage, D., ilk^"^^^^^, s., and senior, A. E. (1988b) Arch, Bwchem. Biophys. 2 6 1 , 222-225 l . Perlin, D. s., cox,D. N., and Senior, A. E. (1983) J. ~ i ~Chem, 258,9793-9800 Pinkham, J. L., and Platt, T. (1983) Nucleic Acids Res. 11, 35313545 Rao, R., Al-Shawi, M. K., and Senior, A. E. (1988a) J . Biol. Chem. 263,5569-5573 Rao, R., Pagan, J., and Senior, A. E. (1988b) J. Biol. Chem 2 6 3 , 15957-15963

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