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Archives of Biochemistry and BiophysicsVol. 363, No. 1, March 1, pp. 135–147, 1999Article ID abbi.1998.1072, available online at http://www.idealibrary.com on

he Extreme Thermostable Pyrophosphatase fromulfolobus acidocaldarius: Enzymatic and Comparativeiophysical Characterization

homas Hansen,* Claus Urbanke,† Veli-Matti Leppanen,‡ Adrian Goldman,‡,§laus Brandenburg,\ and Gunter Schafer*,1

Institute of Biochemistry, Medical University of Lubeck, Ratzeburger Allee 160, D-23538 Lubeck, Germany; \Researchenter Borstel, 23845 Borstel, Germany; ‡Centre for Biotechnology, FIN-20251 Turku, Finland; §Department ofiochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland; and †Institute ofiophysical Chemistry, Medizinische Hochschule Hannover, Hanover, Germany

eceived September 17, 1998, and in revised form December 7, 1998

mPptDf

aeprtsbesfotscalietdoam

Recombinant pyrophosphatase from the hyperther-ophilic archaebacterium Sulfolobus acidocaldarius

S-PPase) has been heterologously expressed in Esch-richia coli and could be purified in large quantities.-PPase, previously described as a tetrameric enzyme,as shown to be a homohexameric protein that had

atalytic activity with Mg21 > Zn21 > Co21 @ Mn21 @i21, Ca21. CD and FTIR spectra demonstrate a similarverall fold for S-PPase and PPases from E. coli (E-Pase) and Thermus thermophilus (T-PPase). The rel-tive proportions of secondary structure elements in-PPase are close to those of a previously proposedodel. S-PPase is extremely heat resistant. Even at

5°C the half-life of catalytic activity is 2.5 h, which isramatically increased in the presence of divalent cat-

ons. More than one Mg21 per monomer is needed foratalysis, but no more than one Mg21 per monomer isufficient for thermal stabilization. The Tm values for-PPase are 89°C (1EDTA), 99°C (1Mg21), and >100°C

1Mn21), compared to 58°C (1EDTA), 84°C (1Mg21),nd 93°C (1Mn21) for E-PPase and 86°C (1EDTA), 99°C1Mg21), and 96°C (1Mn21) for T-PPase. The guanidiumydrochloride-induced unfolding follows an unknownechanism with a biphasic kinetic and an unstable

ntermediate. Unfolding curves of the S-, E-, and T-Pase are independent of the method applied (CDpectroscopy and fluorescence) and show a sigmoidalnd monophasic transition, indicating a change inlobal structure during unfolding, which can be de-cribed by a two-state process comprising dissociationnd denaturation of the folded hexamer into six mono-

1

oTo whom correspondence should be addressed. Fax: 1149-451-

004068. E-mail: schaefer@physik.mu-luebeck.de.

003-9861/99 $30.00opyright © 1999 by Academic Pressll rights of reproduction in any form reserved.

ers. The respective DGN3D(25°C) values of the threePases vary from 220 to 290 kJ/mol for the overallrocess and are not significantly higher for the twohermophilic PPases. The stabilizing effect of Mg21

DG(25°C) is 16 kJ/mol for E-PPase and 5.5–8 kJ/molor S-PPase and T-PPase. © 1999 Academic Press

Cytosolic inorganic pyrophosphatases (EC 3.6.1.1.)re ubiquitously present in all living organisms as anssential catalyst in cellular bioenergetics (1–3). Pyro-hosphatases catalyze the hydrolysis of inorganic py-ophosphate (PPi) into two orthophosphates, thus func-ioning as a thermodynamic trap in coupled reactionshifting the equilibriua of pyrophosphate-generatingiosynthetic reactions toward product formation. Gen-rally, the catalysis is diffusion controlled (4) andtrictly dependent on divalent cations, with Mg21 con-ering the highest efficiency (5). Moreover, Mg21 andther divalent cations protect pyrophosphatase againsthermal denaturation (6, 7). Cytosolic pyrophosphata-es have been isolated from many procaryotic and eu-aryotic sources. Structural and multiple sequencelignments reveal a group of 14 conserved, mostly po-ar, amino acid residues (8, 9). The crystal structures ofnorganic pyrophosphatases from Saccharomyces cer-visae (10, 11), Escherichia coli (9, 12), and the ex-remophilic eubacterium Thermus thermophilus (13)isplay a highly conserved core structure containing allf the invariant residues that are essential for metalnd substrate binding despite large differences in pri-ary structure, molecular weight of the monomer, and

ligomeric structure.

135

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E

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Cadnagmf

tTCls5aFmwAficct

tafiLtpbe

ttppp

dTcsDewsrpsbdet

smapimd

aPnsctaF

136 HANSEN ET AL.

The first inorganic pyrophosphatase from thercheal domain characterized was from Thermoplasmacidophilum (14, 15), a moderately thermophilic or-anism. Recently, we described the isolation, purifica-ion, and cloning of pyrophosphatase from the ar-haeon Sulfolobus acidocaldarius (16), a hyperthermo-hilic organism growing optimally at temperaturesrom 75 to 85°C in an acidic environment (pH 2–3) (17).everal cytosolic proteins from S. acidocaldarius areresently studied with respect to their extreme ther-ostability (18–20).Thermostability and its structural basis in proteins

as been extensively discussed (21–23). The higherhermostability of the T. thermophilus pyrophos-hatase compared to that of the E. coli enzyme washown to be mainly due to larger monomer–monomernterfaces, including both hydrophilic and hydrophobicomponents at the oligomeric contacts (24). However,ctivity-based phenomenological thermostability of py-ophosphatases has never been correlated to thermo-ynamic data and/or unfolding pathways.The difference in free enthalpy between the folded

nd the unfolded state of a protein DGN3D2 is margin-

lly small; for globular proteins, it is between 0.2 and.4 kJ/(mol z residue) (25, 26). Furthermore, the in-rease in DGN3D between homologous thermophilic andesophilic proteins is also quite small (27). Attemps to

btain thermodynamic information on hyperthermo-hilic proteins have often been hampered by the irre-ersibility of the thermal unfolding (28).In our present work we describe the purification of

he recombinant Sulfolobus pyrophosphatase (S-Pase). These preparations as well as pyrophosphata-es from E. coli (E-PPase) and T. thermophilus (T-Pase) were used for spectroscopic assessment of sec-ndary structure as well as biophysical investigation ofhemical- and temperature-induced unfolding. For therst time comparative stability studies are reported foryrophosphatases.

XPERIMENTAL PROCEDURES

Enzymes. The pyrophosphatases from E. coli (E-PPase) and T.hermophilus (T-PPase) were kind gifts of R. Lahti (Department ofiochemistry, University of Turku, Finland) and D. H. Gelfand

Roche Molecular Systems, Inc., Alameda, CA). Their cloning, ex-ression, and purification were described previously (29, 30). Expres-ion of the S. acidocaldarius ppa gene in E. coli and purification werearried out as described (16, 20).

2 Abbreviations used: S-PPase, pyrophosphatase from Sulfolobuscidocaldarius; E-PPase, pyrophosphatase from Escherichia coli; T-Pase, pyrophosphatase from Thermus thermophilus; GdnHCl, gua-idinium hydrochloride; N, native state of a protein; U, unfoldedtate of a protein; I, intermediate state of a protein; K, equilibriumonstant of protein unfolding; xf, fraction of folded protein; xuf, frac-ion of unfolded protein; DGN3D, free enthalpy between the folded

th

nd the unfolded state of a protein; sd, standard deviation; FTIR,ourier transform infrared; DTGS, deuterated triglycine sulfate.

Analytical assays. The purity of the preparations was checked byoomassie brilliant blue R 250-stained SDS–PAGE in 15% gelsccording to standard procedures (31). Protein concentrations wereetermined using the DC protein assay (Bio-Rad, Munich, Germa-y). Analytical gel permeation chromatography was carried out atmbient temperature on a Bio-Sil SEC 250 column equipped with auard column (BioRad; 50 mM Tris–HCl, 150 mM NaCl, pH 7.5; 1l/min). The Phast System (Pharmacia, Uppsala, Sweden) was used

or the determination of the IEP.Analytical ultracentrifugation. For analytical ultracentrifuga-

ion the S-PPase was equilibrated in a buffer containing 50 mMris–HCl, pH 7.0, 5 mM MgCl2, and GdnHCl varying from 0 to 6 M.entrifugation was done at 20°C in a Beckman Optima XL-A ana-

ytical ultracentrifuge equipped with a Titan AN 50 rotor and ab-orption optics. Sedimentation velocity experiments were done at0000 rpm and sedimentation–diffusion equilibrium runs were donet 14,000, 16,000, and 20,000 rpm in a 150-ml volume using 50 mlluorinert FC-43 (ABCR, Karlsruhe) as artificial bottom. When theeasured concentration profile remained unchanged for at least 12 he assumed equilibrium to be attained. The program packageKKUPROG (32) was used to calculate apparent molar masses bytting the ideal distribution for a single species (33) to the measuredoncentration profiles. The partial specific volume of the protein wasalculated from amino acid composition with no correction made forhe influence of guanidinium.

In sedimentation velocity experiments, the apparent sedimenta-ion coefficient sapp

20,W and an approximate molar mass were evalu-ted from the velocity and shape of the sedimenting boundary bytting the time-dependent concentration profiles calculated withamm’s differential equation (34) for a single sedimenting species tohe measured data. The calculations were done using the programackage AKKUPROG (32). Since the shape of the sedimentingoundary is distorted by small heterogeneities of the sample, thisvaluation will yield a lower limit of molar mass.Enzyme assay. Enzymatic characterization including modifica-

ions of buffer composition was carried out as described (14) excepthat the assay temperature was 75°C and pH 7.0. One unit ofyrophosphatase was defined as 1 mmol of pyrophosphate hydrolyzeder minute. Activity data comprise the mean of at least three inde-endent measurements.FTIR spectroscopic measurements. S-PPase and E-PPase were

iluted to a concentration of approximately 40 mg/ml in 20 mMris–HCl, pH 7. Moreover, lyophilized S-PPase was diluted in bufferontaining D2O. Infrared spectra were recorded using a Bruker FTIRpectrometer IFS-55 (Bruker, Karlsruhe, Germany) equipped with aTGS detector. The protein solutions were placed in a cuvetteqipped with CaF2 windows. The pathlength was 8 mm. Spectraere recorded at a nominal resolution of 4 cm21. In order to compen-

ate for H2O absorbance the buffer solutions were measured sepa-ately and subtracted from the protein absorbance. The resultingrotein spectra were deconvoluted as reported (35). Fitting of thepectra was performed with Gaussian band profiles using the Leve-erg–Marquart procedure. Amide I components were assigned toifferent secondary structure types according to theoretical consid-rations and spectra–structure correlations established experimen-ally (35–37).

Thermal stability. For the investigation of long-term thermaltability, pyrophosphatase was incubated at a concentration of 0.1g/ml in buffer containing 25 mM Tris–HCl, pH 7.0, at 75, 80, 85, 90,

nd 95°C, respectively. Either 5 mM MgCl2 or 5 mM EDTA wasresent. Aliquots were taken at different time intervals, stored once, and diluted, and remaining pyrophosphatase activity was deter-

ined. Temperature gradient experiments monitored by circularichroism were performed on a Jasco J 500A spectropolarimeter. The

emperature of the sample was controlled with a Peltier heated cellolder with an incorporated closeable cuvette (V 5 300 ml, d 5 0.5

cpcrscp

sstw

tmolHcPaopSLcTlwe

wflp(vtDe

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R

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C

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m

137PYROPHOSPHATASE FROM Sulfolobus acidocaldarius

m). The protein concentration was set to 100 mg/ml in 20 mM Hepes,H 7.0 (Tm determination) with either 5 mM EDTA or divalentations unless otherwise stated. The temperature of the samples wasaised at a rate of 30°C per hour from 40 to 100°C unless otherwisetated. Protein unfolding was followed by the temperature-inducedhanges of a-helical ellipticity at 226 nm. The fraction of unfoldedrotein was calculated as reported (26).Spectra were recorded in 0.1-cm cuvettes and corrected for the CD

ignal of the solvent; the effective band width was 1 nm. Spectra ofamples as well as solvents were measured 10 times and averaged;he resulting spectra were essentially the same as those in pureater.GdnHCl-induced unfolding. Protein unfolding was detected ei-

her by measuring the changes of a-helical ellipticty at 226 nm or byeasuring the intrinsic protein fluorescence intensity as a function

f GdnHCl concentration. For all samples 10 signals were accumu-ated and averaged. Protein samples were diluted in 50 mM Tris/Cl, pH 7.0, and GdnHCl in 50 mM Tris/HCl was added to a final

oncentration of between 0 and 6 M. Incubation time was 20 h.rotein concentrations varied between 0.04 mg/ml and 0.68 mg/mlnd incubation temperatures were 25 and 50°C. S-PPase containsne tryptophan, and E-PPase and T-PPase contain two trytophanser monomer. Fluorescence measurements were performed using ahimadzu Spektrofluorometer RF 1501 and a Perkin–Elmer LS 50Buminescence Spectrometer equipped with temperature-controlledell holders and were corrected for the solvent fluorescence signal.he optical pathlength of the cells was 1 cm. The excitation wave-

ength in all experiments was 284 nm, and emission was recordedith 90° geometry at 340 nm. The bandwidths for excitation andmission were 2 nm.The fraction of folded protein at a given GdnHCl concentration (xf)as calculated by the use of the respective signal (f) (ellipticty oruorescence intensity) and the corresponding signal of the com-letely folded state (ff) and the completely unfolded state (fuf): xf 5f-fuf)/(ff-fuf); the standard deviations (sd) for the calculated xf

alues were 64–5%. To limitate possible errors the xf values in theransitional region were used for calculation of K(GdnHCl) andGN3D(GdnHCl) only. DGN3D(H2O) values were obtained by linearxtrapolation as described (38).Fluorescence stopped-flow unfolding kinetics were performed at

5°C in a modified version of a Durrum–Gibson stopped-flow appa-atus and analyzed as described in Ref. (39). As stock solutions 0.2g/ml S-PPase and 8 M GdnHCl in 50 mM Tris–HCl and 5 mMgCl2 were used. Final GdnHCl concentrations varied between 4

nd 6.4 M.

ESULTS

Enzyme purification. The purification protocol al-owed us to prepare pyrophosphatase from S. acidocal-arius in high purity and large quantity, sufficient foretailed biophysical investigations. The specific activ-ty in overproducing E. coli cells is more than 100 timesigher than that in the cytosolic extract of S. acidocal-arius. The major purification step, a 4-h treatment at0°C, makes use of the high thermal stability of thenzyme to remove about 89% of the total protein withn eightfold enrichment in activity. The subsequenthree purification steps, ion-exchange chromatographynd two heat treatments at 70 and 80°C (with 5 mMDTA), removed all residual proteins, including con-

aminant E-PPase. Finally, gel filtration served to ob-ain a homogenous preparation of S-PPase exhibiting a

pecific activity of 860 U/mg at 75°C. The yield of pure y

nzyme was 100–200 mg/liter of culture volume; theurified protein ran as a single band on SDS–PAGEnd on isoelectric focussing with an isoelectric point of.8 (Table I) demonstrating the slightly acidic charac-er of the Sulfolobus pyrophosphatase. The identity ofhe recombinant protein with the enzyme from S. aci-ocaldarius could be verified by immunoblotting.Oligomeric structure. In our previous work the py-

ophosphatase from S. acidocaldarius had been re-orted to be a homotetramer of 71 6 8 kDa. Whenssayed by analytical gel filtration, this behavior coulde confirmed for the recombinant protein as well. Theame was also suggested by analysis of sedimentationata. Figure 1 shows a sedimentation velocity experi-ent with S-PPase giving a sedimentation coefficient

f 6.59 6 0.16 S. The shape of the sedimenting bound-ry is best described by a molecular mass of 82 6 30Da using a partial specific volume of 7.42 1024 m3/kg.ince for a perfect unhydrated sphere of 82 kDa theedimentation coefficient was calculated to be 6.8 S,his result suggests that the protein is at least tet-americ but does not exclude a larger oligomerization.igure 2 shows a sedimentation diffusion equilibriumxperiment in absence and presence of 3 M GdnHCl,

TABLE I

Major Properties of the Pyrophosphatasefrom Sulfolobus acidocaldarius

R 19365a

amino acids 173a

ligomeric state Hexamericb

EP 4.8H optimum 7.0c

ubstrate specificity Pyrophosphate (100%), pNP,PEP (1–2%); ATP, P3,ADP (3–6%); TTP, ITP (10%)c

ation specificity (5 mM) Mg(100%) . Zn(95%) .Co(88%) @ Mn(32%) @Ni(3%), Ca(2%)c

pecific activity (75°C, pH 5 7) 860 U/mgc

m 5.4 6 0.4 mMc

urnover 1700 s21c

cat/Km 2 3 108 M21 s21

M, Mg 0.9 mMc

nhibitors F2, Ca21, phenylglyoxalH stability 4.5–9c,d

emperature-optimum 75°Ce

a 80 kJ/molf

1/2 at Topt .24 h at 75°Cc

tabilizing ions Mg21, Co21, Mn21, Zn21, Ni21

a MR, n of monomers; from sequence data (16).b As judged by equilibrium ultracentrifugation.c These data represent an average of at least three independenteasurements.d As judged by CD spectroscopy.e Incubation time 20 min (16).f Taken from (16).

ielding molecular masses of 114 6 5 and 23 6 1 kDa,

rhFPsca

ttsmpplssCwthit

csbcy1i

oevtopwbte

Sta2oTmr

paHhbbTPm

Fr repa

138 HANSEN ET AL.

espectively. This clearly indicates a transition from aexamer to a monomer upon GdnHCl denaturation.igure 3 shows the apparent molecular mass of S-Pase as a function of GdnHCl concentration. Thelight increase in molecular mass at high GdnHCl con-entrations possibly is a result of preferential denatur-nt binding not accounted for in the evaluation.Enzymatic characterization. The main characteris-

ics of S-PPase are listed in Table I. Under the condi-ions tested, the pH optimum was 7.0 6 0.1. S-PPasehows a pronounced preference for PPi, which wasore readily cleaved than any other substrate. Phos-

hoester bonds were cleaved more slowly than phos-hoanhydride bonds (Table I). The enzyme is abso-utely dependent on divalent cations with Mg21 (100%)howing the highest activity, which can be efficientlyubstituted by Zn21, Co21, and Mn21. Neither Ni21 nora21 was able to activate the enzyme for catalysis. Asith other pyrophosphatases (4), Ca21 even inhibits

he cleavage of PPi in the presence of 5 mM Mg21;owever, inhibition was weak and no more than 40% of

nhibition could be achieved at high Ca21 concentra-ions (50 mM).

Kinetic parameters as summarized in Table I indi-ate that the hydrolysis of pyrophosphate is limitedolely by diffusion. The KM for Mg21 was determined toe 0.9 mM. Obviously, Mg21 serves as more than just aomplexing ion for pyrophosphate. Further data anal-sis of the Mg21 effects resulted in a Hill coefficient of.9, suggesting that more than one Mg21 participates

IG. 1. Sedimentation velocity experiment of S. acidophilus pyrophpm. Traces were scanned in intervals of 390 s. The smooth curvesmolecular mass of 82 6 30 kDa and 6.59 6 0.16 S.

n the catalytic mechanism, similar to other PPases (8). s

NaF (1.7 mM) inhibits S-PPase by about 50%. More-ver, the inhibitory effect of phenylglyoxal, which pref-rentially modifies arginine residues, was used to in-estigate the possible involvement of these residues inhe catalytic process. With 5 mM phenylglyoxal, webserved 50% inhibition after 3 min and almost com-lete inhibition after 20 min incubation at 75°C,hereas addition of 2 mM pyrophosphate in the incu-ation solution protected S-PPase against this inhibi-ory effect, which was reduced to only 10% loss ofnzymatic activity.Circular dichroism spectroscopy. The CD spectra of

-PPase showed one negative peak at 207 nm. In con-rast, both E-PPase and T-PPase each display two neg-tive peaks at 209 and 220 nm (Fig. 4) and at 207 and21 nm, respectively (Fig. 4). Otherwise, there werenly slight differences in the CD spectra of S-, E-, and-PPase, with the exception of a much higher maxi-um around 193 nm for the Thermus enzyme. This

eflects an increased fraction of helical structure.Resolution-enhanced FTIR spectra of pyrophos-

hatases. Figure 5 shows the infrared spectra in themide I region of the S-PPase and E-PPase dissolved in

2O. An analysis with the aid of a band-fitting routineas been performed and the resulting componentands are presented in the same figure. A summary ofand positions as well as area fractions is given inable II. The absorption spectra of S-PPase and E-Pase in H2O showed a very similar pattern with aaximum at 1651 cm21 and broad shoulders on both

hatase in 0.05 M Tris, pH 7.0, 5 mM MgCl2 run at 20°C and 500,000resent a theoretical sedimenting boundary for a single species with

osp

ides consisting of six components, suggesting compa-

rtwDa

tctim

pv

nwmvteM

F(r

139PYROPHOSPHATASE FROM Sulfolobus acidocaldarius

able backbone structures in the two enzymes. Three ofhe six band positions were identical for both enzymes,hereas the other three display minor differences. In2O the absorbance peaks of the S-PPase were sharpernd in most cases shifted to lower values.The FTIR spectra allow a quantitative estimation of

he secondary structure by the assignment of amide Iomponent bands to different types of secondary struc-ure elements. This assignment of amide I componentss given in Table II and the calculated structural ele-

IG. 2. Equilibrium analytical centrifugation of S. acidophilus pyroB) 3 M GdnHCl. The smooth curves represent theoretical concentrespectively. The “noisy” curves show the residuals of the fit.

ents are compared in Table III. For the two pyrophos- m

hatases investigated, the FTIR analysis reveals aery similar secondary structure.Thermostability. S-PPase displayed an extraordi-

arily strong long-term heat resistance up to 95°Chich was dramatically enhanced in the presence of 5M Mg21 (Figs. 6A and 6B). The half-times of inacti-

ation were 2.5 h (1EDTA) and 6 h (1Mg21), respec-ively. Following 8 h of incubation at 85°C, 30% ofnzymatic activity was retained without, but 95% with,g21 present. S-PPase underwent practically no ther-

osphatase in 0.05 M Tris, pH 7.0, 5 mM MgCl2 with (A) and withouton distributions for molecular masses of 114 6 5 and 23 6 1 kDa,

phati

al inactivation at 75°C without and at 80°C with

Mam

P

tmsrf

F atac

Fpo

140 HANSEN ET AL.

g21, respectively. The rates of enzymatic inactivationt 80 and 85°C were also paralleled by changes in CDeasurements of a-helical ellipticity (not shown).Furthermore, the unfolding of the S-, E-, and T-

Pase was monitored by a-helical ellipticity as a func-

IG. 3. Apparent molecular mass of of S. acidophilus pyrophosphoncentrations of guanidinium chloride.

IG. 4. CD spectra of the pyrophosphatases from S. acidocaldarius

H 7.0, and the protein concentration was set to 100 mg/ml. Each spectf 10 measurements.

ion of temperature (Figs. 7A–7D). The sigmoidal andonophasic melting curves were consistent with a two-

tate mechanism. The resulting values for Tm wereeproducible within 62 and 58°C for the E-PPase, 86°Cor the T-PPase, and 89°C for the S-PPase, respec-

se in 0.05 M Tris, pH 7.0, 5 mM MgCl2 denatured with different

. coli, and T. thermophilus. Spectra were recorded in 20 mM Hepes,

, E rum was corrected for the solvent signal and represents an average

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141PYROPHOSPHATASE FROM Sulfolobus acidocaldarius

ively. In the presence of Mg21 the Tm values werepshifted to 84°C for the E-PPase and increased to9°C for the S- and T-PPase (Table IV). One Mg21 peronomer was found to be sufficient for introduction of

he enhanced thermal stability, whereas no effect at allould be detected with 0.5 Mg21 per monomer. In com-arision, Mn21 exerted an even more dramatic stabi-izing effect in the E-PPase and S-PPase, but not in-PPase. The Tm values were enhanced to 93°C in the-PPase and to 96°C in the T-PPase; however, the-PPase did not show any unfolding at all up to 100°C

higher temperature were not tested).While long-term incubation near 100°C caused ag-

regation as judged by an increase in light scattering,ooling on ice immediately after reaching the highestemperature (e.g., 100°C) in the applied heating profilenduced refolding of the three PPases with an 80–90%

IG. 5. Fourier self-deconvoluted amide I band contours of pyro-hosphatases from S. acidocaldarius (A) and E. coli (B) in H2O. Thepectra have been analyzed with the aid of band-fitting routine (cf.aterial and Methods), and the resulting component bands are

epicted in the same figure.

ecovery of native structure. An additional 16 h at 4°C

ed to further recovery of secondary structure. Consec-tive temperature gradients of S-PPase with an inter-ediary 15-min cooling period are depicted in Fig. 7B.

nterrupting the gradients at the respective Tm allowedlmost complete refolding. Moreover, the reversibilityf thermal unfolding of the S-PPase has also beenonfirmed by FTIR spectroscopy. After 1 h at 100°C noecondary structure was detectable, but rapid coolingestored the initial spectra within 10–15 min (Fig. 5A).The Tm value of the Sulfolobus pyrophosphatase was

ndependent of the temperature gradient applied in aange of 0.25–1°C/min, indicating that irreversibleteps like aggregation were rather slow. The calculatedGN3D(Tm) values are summarized in Table IV. Study-

ng the effects of a variety of divalent cations the fol-owing stabilizing order was established: Ni21 (96°C) ,

g21 > Co21 > Zn21 (98°C) , Ca21 (102°C, extrapo-ated) , Mn21 (@100°C).

GdnHCl-induced unfolding. Unfolding of S-PPaseediated by the chaotropic reagent GdnHCl has been

tudied by analytical ultracentrifugation, fluorimetry,nd CD spectroscopy. As shown in analytical equilib-ium ultracentrifugation (Fig. 3), the transition fromexamers to monomers occurs in the range between 2nd 3 M GdnHCl. Although the protein concentrationradients in the transition region clearly showed ateast two species with different molecular masses to beresent, a quantitatve interpretation was impossibleue to the inherent uncertainties of the method. Nev-rtheless, stopped-flow monitoring of unfolding kinet-cs by endogenous fluorescence could be fitted only by aiphasic process, indicating a short-lived unstable in-ermediate. At 4.2 M GdnHCl data were best fittedith t1 5 49 s and t2 5 511 s at 4.2 M GdnHCl, whereast .6.4 M GdnHCl, only a monophasic process with t 5s could be resolved.Figure 8 demonstates the GdnHCl dependence of

nfolding of S-, T-, and E-PPase as monitored by inter-al protein fluorescence. In accordance with a two-tate model of simultaneous dissociation and unfoldingf a native hexamer (N) into six unfolded monomersU), the fraction of folded protein (xf) and therefore thenflection point of the transition curve was dependentn the initial protein concentration (c0) as explicitelyemonstrated here for S-PPase (Fig. 8).

NN 6U [1]

K 5c U

6

cN[2]

ith cN 5 c0 z xf and cU 5 6(1 2 xf) z c0

6 6~1 2 x f!6c0

5

K 5 x f[3]

cgdcsM

caTfsapfoGexptdstnimhpcnvlTc

ekv(

D

bppPa

F

X

M

142 HANSEN ET AL.

In accordance with Eq. [3] the effect of the proteinoncentration on xf is obvious in the transitional re-ion, where the fraction of folded protein xf showed aramatic increase (Fig. 8) upon raising the proteinoncentration from 0.04 to 0.68 mg/ml. It resulted in ahift of the GdnHCl titration curve to the right by 0.3

GdnHCl.Further studies of GdnHCl-induced unfolding in-

luded E-PPase and T-PPase. Corresponding sigmoidalnd monophasic behavior was observed with E- and-PPase irrespective of the method used to monitor the

olded state, fluorescence, or CD spectroscopy (nothown). The evaluated data are compiled in Table V. Insimple two-state process (unfolding of a monomeric

rotein) DGN3D(H2O) is related to the midpoint of un-olding (xf 5 0.5) and to the slope m taken from a plotf the calculated DGN3D(GdnHCl) values versusdnHCl concentrations, which is used for the linearxtraploation of DGN3D(H2O) at GdnCl 5 0. However,f 5 0.5 as well as m are dependend on the initialrotein concentration for unfolding of oligomeric pro-eins like the hexameric S-PPase (Table V). Due to theifferent slopes of the corresponding curves in the tran-itional region, an increase in the GdnHCl concentra-ion at the midpoint of the unfolding curves did notecessarily reflect an increased thermodynamic stabil-

ty at 0 M GdnHCl. This explains why S-PPase wasore stable than the E. coli enzyme even though it wasalf-unfolded at lower GdnHCl concentrations. In theresence of 5 mM EDTA at 25°C, the mean free energyhange for the overall process of dissociation and de-aturation was DGN3D(H2O) 5 243 kJ/mol (meanalue) for the Sulfolobus enzyme, whereas the equiva-ent values for E-PPase were 23 kJ/mol lower and for-PPase 39 kJ/mol higher (mean value). Again, the

TAB

Positions of Amide I Component Bands, Relative Integfor Pyrophosphatase from

S. acidocaldarius

D2O H2O

Band position(cm21)

Band area(%)

Band position(cm21)

Band a(%)

1608 13 1616 111628 31 1634 221644 171659 22 1651 28

1668 231675 16

1683 151692 1 1694 1

ontribution of the stabilizing effect of Mg21 was high-

st for E-PPase corresponding to a DGN3D(25°C) of 16J/mol (mean value) compared to 5.5 (25°C, meanalue) and 7 kJ/mol (50°C) for S-PPase and 8 kJ/molmean value) for T-PPase, respectively.

ISCUSSION

Oligomeric structure. Pyrophosphatases from eu-acterial and archebacterial sources have been re-orted to be tetrameric or hexameric enzymes com-osed of subunits of about 20 kDa. In contrast to thePase from T. acidophilum (14), gel filtration on annalytical column constantly suggested a tetramer,

II

ted Intensities, and Secondary Structure Assignmentacidocaldarius and E. coli

E. coli

Assignment

H2O

Band position(cm21)

Band area(%)

1618 24 b-Sheet1634 18 b-Sheet

Unordered1651 25 a-Helical, unordered1668 20 Unordered

b-Turn, b-Sheet1682 12 b-Turn, b-Sheet1695 1 b-Sheet

TABLE III

Comparison of Secondary Structural Data from X-Ray,FTIR, and Modeling for the Pyrophosphatases from

S. acidocaldarius, E. coli, and T. thermophilus

Enzyme S. acidocaldarius E. coli T. thermophilus

TIR 51% a 1 u 45% a 1 u n.d.49% b 1 bt

(H2O)55% b 1 bt

-raycrystallography 16.0% aa 16.7% ab

30.3% b 35.1% b9.7% bt 6.3% bt44.0% u 33.3% u

8.6% 310

odel 16.2% ac

34.1% b7.5% bt42.2% u

Note. n.d., not determined.a As taken from PROCKECK output (43).b

LE

raS.

rea

As taken from PROCHECK output (11).c Taken from an energy minimized model (16).

wrsp4rtsP(cw

am

tpsSMaafrwlPcHssa4(mswi

mhbcaadmbbssaswt

oahETcmbct

Fcp(8wai

143PYROPHOSPHATASE FROM Sulfolobus acidocaldarius

hile sedimentation equilibrium ultracentrifugationevealed a hexameric structure. The observed effectuggests a very tight packing of the oligomer and ap-lies equally to the adenylate kinase of Sulfolobus (40,1) as well as to the E. coli enzyme. Since all “tet-americ” PPases were determined either by gel filtra-ion or sedimentation ultracentrifugation, we expectimilar corrections of Mr also to be necessary for thePases from Methanobacterium thermoautotrophicum

strain DH) (42), Sulfolobus sp. strain 7) (43), Thioba-illus thiooxydans (44), and Thermus aquaticus (45),

IG. 6. Long-term stability of the pyrophosphatase from S. acido-aldarius. The protein was diluted in 25 mM Tris–HCl, pH 7.0, in theresence of 5 mM EDTA (A) as well as in the presence of 5 mM Mg21

B) to a final concentration of 100 mg/ml. Symbols: (■) 75°C, (F)0°C, (Œ) 85°C, (�) 90°C, and (}) 95°C. Curve fitting was performedith linear regression as well as exponential first-order decay as anpproximation. Each data point represents the average of threendependent measurements with mean sd values of 64.

hich had been classified as homotetramers. Presum- 5

bly a homohexameric oligomeric structure is the nor-al case for all eubacterial and archebacterial PPases.Enzymatic characterization. The catalytic proper-

ies of the S-PPase resembled those of the cytosolicyrophosphatases known so far, except that the cationpecifity is less pronounced (Table I). In contrast to-PPase, the relative effectiveness in comparision tog21 was only 6% for Co21 and Zn21 for E-PPase (46)

nd 37% (Co21) and 21% (Zn21) for the PPase from T.cidophilum (14). However, Mg21 exerts multiple ef-ects on PPases. In S-PPase one high-affinity site (mMange) is responsible for protein stabilization (Fig. 7A),hile at other sites Mg21 acts as an activator and a

igand for MgPPi binding as clearly demonstrated forPases from E. coli and S. cerevisae (47, 48). This isorroborated by the apparent KM 5 0.9 mM for Mg21, aill coefficient of 1.9 for Mg-dependent activation. Be-

ides Mg21, an invariant Arg residue contributes toubstrate binding. According to previously reportedlignments Arg 40 of the S-PPase corresponds to Arg3 of E. coli and Arg 78 of S. cerevisae, respectively16). These arginines are known from solution studies,

utagenesis, and x-ray structures to be involved inubstrate and product binding (4, 11, 46). This is in lineith the protective role of PPi against the phenyglyoxal

nhibition observed here.Structural characterization. The three-dimensionalonomeric structures of E-PPase and T-PPase

ave a very similar globular fold (11, 24). The resem-lance of both CD and FTIR spectra of the S-PPaseompared to those of the E-PPase and T-PPase indicatevery similar secondary structure pattern and hence

re strong arguments in favor of our previously pre-icted model of S-PPase (16). The FTIR-derived assign-ent of secondary structure elements shown here can

e validated on that basis (Table III). In both cases theand at 1668 cm21 has been assigned to unorderedtructures. Although this assignment is based on thehift of this band to 1644 cm21 in D2O (37), absorptiont 1668 cm21 might be partially due also to b-turntructures. The fraction of b-sheet 1 b-turn structuresas overestimated by 15% for E-PPase and based on

he S-PPase model by 7.4% for the S-PPase.The global structure was not changed by the addition

f Mg21. The CD spectrum in the UV region of the S-nd T-PPase were not affected on addition of Mg21 asas also been reported for the PPases from S. cerevisae,. coli (7), PS 3, and Bacillus stearothermophilus (6).he fluorescence intensity of E-, S-, and T-PPase in-reased by 4–7% indicating only rather subtle confor-ational changes which did not alter the polypeptide

ackbone. This is consistent with the lack of structuralhange seen on going from the apo-E-PPase (9, 12) tohe magnesium- or manganese-containing forms (49,

0).

prtqpo(iolaeMvw1Msc

1walt

Fptt .5°

SE

144 HANSEN ET AL.

Thermotolerance and protein stability. Whereasrevious studies of the thermostability of PPases haveelied on determination of heat resistance and catalyticemperature optima, the present approach exploitsuantitative data on the fraction of folded and unfoldedrotein as a function of temperature. Accordingly, thenset of unfolding as monitored by CD spectroscopyT1) correlates with the sharp decline of catalytic activ-ty several degrees above the catalytic temperatureptima. At the very same temperature the property ofong-term stability is simultanously lost. This methodlso allows a more precise differentiation of stabilizingffects, for example, by divalent cations. In each caseg21 and Mn21 caused a significant upshift of the Tm

alues. This shift was most dramatic for the E-PPaseith a 126°C increase versus 13°C for T-PPase and0°C for S-PPase, respectively. While the effects ofg21 and Mn21 were comparable for T-PPase, Mn21

hifted the T from 58 to 93°C for E-PPase and in the

IG. 7. Examples of thermal-induced unfolding experiments as relotted as a function of temperature. Pyrophosphatases from S. ahermophilus (D). Samples were diluted in 20 mM Hepes, pH 7.0, wo 100 mg/ml. The temperature was increased at a constant rate of 0

m

ase of S-PPase prevented any unfolding up to at least T

00°C. Although it has not been proven in detail, oneould predict that the catalytic temperature optimare shifted accordingly. However, the different stabi-izing effects of divalent cations did not correlate withheir catalytic efficiency when measured under stan-

ded by CD spectroscopy. The fraction of unfolded protein (xuf) wasocaldarius (A 1 B), consecutive gradients (B), E. coli (C), and T.5 mM EDTA, Mg21, or Mn21 present. Protein concentration was setC per minute. T1 gives the onset of unfolding.

TABLE IV

Measured Tm Values and Calculated Values for DGN3D forthe Pyrophosphatases of S. acidocaldarius, E. coli, and

T. thermophilus at the Respective Tm (The Reproducibilityof the Tm Was about 62°C)

Enzyme

Tm [°C]/DGN3D (H2O) (kJ/mol)

1EDTA(5 mM)

1Mg21

(5 mM)1Mn21

(5 mM)

ulfolobus 89/188 98/193 .100./195. coli 58/172 84/185 93/190

Additive:

corcidith

hermus 86/186 99/194 96/192

doc

Fb ctp l (}s

t

145PYROPHOSPHATASE FROM Sulfolobus acidocaldarius

ard assay conditions. Our findings contradict previ-us studies on E-PPase which reported an equal effi-iency for Mn21 and Mg21 as well as a cation concen-

IG. 8. GdnHCl-induced unfolding of the pyrophosphatase from S.y fluorescence. The fraction of folded protein (xf) was plotted as a funH 7.0, 5 mM MgCl2. The protein concentration was set to 0.68 mg/md values of the data were 4%.

TAB

Analysis of GdnHCl-Induced Unfolding of the Pyrophosphas Monitored by CD Spectroscopy (C

T(°C) Additive Signal

cPPase

(mg/ml)

S. acido

25 Mg21 FI 0.68Mg21 FI 0.04EDTA FI 0.04EDTA CD 0.04

50 Mg21 CD 0.17EDTA CD 0.12

E.

25 Mg21 FI 0.04Mg21 CD 0.04EDTA FI 0.04

T. ther

25 Mg21 FI 0.03EDTA FI 0.03EDTA CD 0.03

Note. xf 5 0.5 was taken from a plot of xf versus GdnHCl concentrathe GdnHCl concentration by linear extrapolation with the slope m to

tration dependence for the cation-induced stabilizationin the mM range (7). As we found Mg21-induced sta-blization in the mM range, it is likely that cation-in-

idocaldarius (F, E), E. coli (■), and T. thermophilus (Œ) as followedion of GdnHCl concentration. Standard buffer was 20 mM Tris–HCl,) and 0.04 mg/ml (■, F, Œ), and incubation time was 24 h. The mean

V

ases from S. acidocaldarius, E. coli, and T. thermophilus) and/or Fluorescence Intensity (FI)

xf 5 0.5 atcGdnHCl (M)

m(kJzlzmol22)

DGN3D(H2O)(kJ/mol)

ldarius

3.2 234 6 4 247 6 112.9 229 6 1 250 6 33.1 225 6 2 246 6 63.1 223 6 1 240 6 22.8 229 6 3 243 6 82.9 224 6 4 236 6 10

li

4.9 214 6 1 236 6 34.9 214 6 2 236 6 64.5 211 6 1 220 6 7

philus

5.1 24 6 2 290 6 64.7 24 6 2 283 6 64.7 24 6 4 281 6 10

ac

LE

atD

ca

co

mo

ion. DGN3D(H2O) was taken from a plot of DG(GdnHCl) as a function ofc(GdnHCl) 5 0 (36).

dfchPopPmogiP

EmeSdlsraatI5xu

stPkTthamDalliknamavwttc

q

a(

HPktcamiiPiE

A

stc

ttaS

R

1

1

1

146 HANSEN ET AL.

uced stabilization is the physiologically normal caseor all three PPases studied. Therefore, E-PPase can belassified as a thermophilic protein in a mesophilicost, whereas the thermostability of S-PPase and T-Pase are typical of proteins from hyperthermophilicrganisms with Tm values close to 100°C (20). Unex-ected thermostability has also been described for thePases from T. acidophilum (14), Bartonella bacillifor-is (51), and Bacillus subtilis (52) with temperature

ptima 20–30°C above the respective physiologicalrowth temperature of these organisms, which mightndicate a thermophilic evolutionary origin for thesePases.From the respective optima of growth temperature of. coli (37°C), S. acidocaldarius (75°C), and T. ther-ophilus (75°C) one would expect an approximately

qual thermostability of the T-PPase compared to the-PPase, which indeed is about the case. Althoughenaturation was followed by irreversible steps, theatter were kinetically unfavored and rather slow; con-equently, a situation quite close to equilibrium waseflected by the unfolding curves. This holds especiallyt the beginning of the curves. Taking Eq. [3] intoccount, the respective values for DGN3D(Tm) of thehree hexameric PPases were still large at Tm (TableV) when compared to monomeric proteins (DGN3D(Tm)

0). In hexameric proteins DGN3D 5 0 corresponds touf > 1 at the end of the transitional region of thenfolding curve.Finally, comparative GdnHCl-induced unfolding

tudies of the three PPases allowed determination ofhe stabilizing contribution of Mg21 in the threePases. The Mg21 induced increases in DGN3D were 16J/mol in E-PPase and 5.5–8 kJ/mol in S-PPase and-PPase. This corresponds to a few additional interac-ions like nonionic hydrogen bonds, salt bridges, orydrophobic interactions, which may vary between 2nd 4 kJ/mol (53, 54) and 20 kJ/mol (21, 55) as deter-ined for other proteins. An average increase ofGN3D(25°C) by Mg21 of 6–8 kJ/mol corresponds to anverage increase in Tm of 10°C. These findings are inine with stability studies on mutants of phage T4ysozyme which revealed an increase of thermostabil-ty of up to 12°C, corresponding to an extra 12–25J/mol (56). A possible explanation of the more pro-ounced effect of divalent cations in E-PPase might bedduced to a different coordination of the tightly boundetal ion, M1 (terminology as in Ref. (11)), in the

ctive site in E-PPase. Comparison of the DGN3D(25°C)alues both in the presence and in the absence of Mg21

ere in the order: E-PPase , S-PPase , T-PPase, withhe PPases from the thermophilic sources, especiallyhe T-PPase, having a slighly higher stability at 25°Compared to the E-PPase.

GdnHCl induced unfolding of S-PPase follows a se-

uential mechanism of dissociation and unfolding with

n intermediate (I) as common for oligomeric proteins57).

NN nI6/nN 6U [4]

owever, the detailed mechanism remains unknown.resumably a short-lived intermediate is formed in ainetically limitating slower phase of complete or par-ial dissociation with rapid subsequent unfolding. Thatould explain why no intermediates were found in thenalytical ultracentrifugation runs. Trimers or mono-ers, in contrast to dimers, are likely candidates for an

ntermediate state of S-PPase dissociation and unfold-ng mechanisms as those were found among some E-Pase variants (58–60) and as a result of the acid-

nduced dissociation of PPases from Bacillus (61) and. coli (62).

CKNOWLEDGMENTS

This work was supported by grants from the Deutsche For-chungsgemeinschaft (Scha125/17-3). We thank Dr. von Busse forhe assistance with the FTIR and Achim Kraus for the help with theomputers.

Note added in proof. While this paper went to press a refinedhermodynamic treatment of thermal unfolding of homo-oligomerichermostable adenylate-kinase appeared which fully supports thebove-described approach for pyrophopsphatases [J. Backmann, G.chafer, L. Wyns, and H. Bonisch (1998) J. Mol. Biol. 284, 817–833].

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