Biochemistry1988,27, 1581-1587

r5 8 l

Reconstructionby Site-DirectedMutagenesisof the Transition State for the Activation of Tyrosine by the Tyrosyl-tRNA Synthetase: A Mobile Loop Envelopesthe Transition State in an Induced-Fit Mechanismt Alan R. Fersht,*'Ï Jack W. Knill-Jones,THugues Bedouelle,$ and Greg Winter$ Departmentof Chemistry,Imperial Collegeof Scienceand Technology,LondonSW7 2AY, (J.K.,and MRC Laboratoryof Molecular Biology, MRC Centre,Hills Road, CambridgeCB2 2QH, U.K. Receiued Junei,5, 1987;Reuised ManuscriptReceiued OctoberI,;,987

ABsrRAcr: Site-directedmutagenesisof the tyrosyl-tRNA synthetasefollowed by kinetic studieshas shown that residueswhich are distant from the active site of the free enzyme are brought into play as the structure of the enzyme changesduring catalysis. Positively charged side chains which are in mobile loops of the enzymeenvelopethe negatively charged pyrophosphatemoiety during the transition state for the formation of tyrosyl adenylatein an induced-fit mechanism. ResiduesLys-82 and A19-86, which are on one side of the rim of the binding site pocket, and Lys-230 and Lys-233, which are on the other side,have been mutated to alanine residuesand also to asparagineor glutamine. The resultant mutants still form 1 mol of tyrosyl adenylate/mol of dimer but with rate constantsup to 8000 times lower. Constructionof differenceenergy diagramsrevealsthat all the residuesspecificallyinteract with the transition state for the reactionand with pyrophosphatein the E.Tyr-AMP.PP1complex. Yet, the e-NH3+groupsof Lys-230 and Lys-233 in the crystalline enzymeare at least 8 A too lar away to interact with the pyrophosphatemoiety in the transition state at the same time as do Lys-82 and A19-86. Binding of substratesmust, therefore, induce a conformational changein the enzymethat brings theseresiduesinto range. Consistentwith this proposalis the observationthat all four residuesare in flexible regionsof the protein. The induced-fit mechanismallows accessof substratesto the active site and enablesthe transition state to be completely surroundedby groups on the protein which would otherwise block entry.

Ihe aminoacyl-tRNA synthetases catalyzethe formation of aminoacyl-tRNA by frrst forming an enzyme-boundaminoacyl adenylatecomplex(eq l, =activation)and then transferring the aminoacid to tRNA (eq2). The mechanismof activation E + AA + ATP = E.AA-AMP + ppi (1) E.AA-AMP + tRNA = AA-IRNA + E + AMP (2) has proved an enigma. Despiteintensivework on the 20 different typesof enzyme,there is no convincingevidencefrom classicalkinetic experimentsor protein chemistry for any uniquelyimportant residuesas usedin conventionalacid-base or covalentcatalysis. Further, there is little convincingsequencehomologybetweensynthetases apart from the motif (numberedaccordingto the tyroCys-35...His-45...His-48 syl-tRNA synthetase)noted first at the active site of the tyrosyl-and methionyl-tRNA synthetases (Barker & Winter, 1982). The initial crystal structuresalso failed to identify catalytic groups,but systematicsite-directedmutagenesishas begun to unravel the catalytic mechanism(Winter et al., 1982). Residuesthat appearto interact with the substrates in the crystalstructureof the enzyme-bound tyrosyl adenylate complexhavebeenmutated. Residuesthat are nominally in the site for binding adenosinehavebeenfound to bind ATP not in the ground state (enzyme-tyrosine-ATP or enzymeATP complexes)but in the transitionstateand enzyme-bound tyrosyladenylatecomplex(e.g.,Cys-35and His-48;Wells & Fersht,1986;Fershtet al., 1986a). Mutagenesis of His-45 indicatedthat it was involvedin binding the transitionstate of the reagents. Subsequentmodel building of the enzymelThis work was supported by the MRC of the U.K. I Imperial College of Scienceand Technology. s MRC Centre.

transition-statecomplexrevealeda binding site for the 7phosphateof ATP betweenThr-40 and His-45in the transition state, which was confirmed by mutagenesisof Thr-40 (Leatherbarrowet al., 1985;Leatherbarrow& Fersht,1987). An important strategyfor analyzingthe resultsof mutation hasbeento determinethe apparentcontributionto the binding energyof the mutatedsidechainat eachstateof the reaction from differenceenergydiagrams(Wells & Fersht,1986;Ho & Fersht,1986). Theseare constructed by comparison of the free energyprofilesof the reactionof wild-type and mutant enzymesas determinedby measuringall the necessaryrate and equilibriumconstantsin eq 3. Differenceenergydiagrams TYt.-_

L. .-

_4JP.__

h,.t yr ;-

Kt'K^'k_,

k3

E.Tvr.ATP ;E.Tyr-AMP.PPi t.trr-AMP ' * PPi

(3)

showdirectly the effectsof the mutation on the binding of each substrate,intermediate,and transitionstate. A spuriouseffect of a residuein the ATP site affecting the binding of tyrosine, for example,is immediatelyobvious. The combinationof sensibledifferenceenergydiagrams,sensitivekinetictests[e.g., the double-mutant test (Carteret al., 1984)],but especially the finding of linear free energyrelationships(Fersht et al., 1986b,1987)and X-ray crystallographic evidence(Brown et al., 1987)providesstrongevidencethat the resultsof these mutationsreflectjust the local effectsof the changeof side chain rather than any grosschangein enzymestructure. BeyondDirect CrystallographicInformation. The kinetic methodshavebecomeevenmore importantat the next stage of the investigationbecausewe havenow reachedthe limits of informationpresentlyavailablefrom proteincrystallography on direct interactionsbetweenthe enzymeand its substrates.

0006-2960 @ 1988AmericanChemicalSociety 18810427-1s81$01.s0l0

1582 BIocHEMISTRY The crystal structuresof the free enzyme and of the enzyme-tyrosinecomplexesare thoseof a symmetricaldimer (Brick & Blow, 1987). But, it is knownfrom solutionstudies that only I mol of tyrosineis boundper moleof dimer (Fersht, 1975) and that binding is accompaniedby a changein the tryptophanfluorescence.Only 1 mol of tyrosyl adenylateis by a further similar change formed,and this is accompanied in fluorescence(Fersht et al., I975). Substrate-induced conformationalchangesare clearlyindicated,but their magnitude in structural terms is unknown. But, could these changesbring residuesdistantfrom the activesitein the crystal structureinto the mechanism?Cluescame from a study in which most of the basic residuesof the enzymehad been mutated to map out the tRNA binding site (Bedouelle& Winter, 1986). This revealedthat severalmutants (Lys Asn-233) Asn-82,Arg - Gln-86,Lys - Asn-230,and Lys----weredefectivein activationof tyrosine. Lys-82 and A19-86 areat one sideof the entranceto the substratebinding pocket and Lys-230and Lys-233 are at the oppositeside. We have now characterizedthesemutantsand a further set in which residues82, 86, 230, and 233 have each been mutated to alanine. PRocnnuRes ExpBRrl{eNTAL Materials Restrictionand other enzymeswere obtainedfrom Boehringer-Mannheim,chemicalsfrom Sigma (London),and radiochemicalsfrom AmershamInternational. Methods Productionof Mutants. Mutants Lys - Asn-82,Arg Gln-86,Lys * Asn-230,and Lys - Asn-233werepreviously constructed(Bedouelle& Winter, 1986)on a derivativeof the originalMl3mp93TyrS vector(Winter et al., 1982)altered in two waysin the TyrS gene: (i) a deletionof 900 bp to the 5'side of the promoter;(ii) deletionof a prematureterminator betweenthe promoterand the start of translation[elements 2 and3 of Waye and Winter (1986)1.This improvesthe yield of TyrTS about 3-fold in infectedcells. For experimental detailsand the strain constructionsusedin the site-directed seeCarter et al. (1985): the mutagenicoligomutagenesis, (Bedouelle& Winter, 1986)wereusedin *double nucleotides priming' with the strand selection primer SELl on Ml3mpg3amIVTyrTS geneIV, transfectingthe heteroduplex into competentEscherichia coli HB2154 with a lawn of H82151. In the presentstudy,mutant Lys * Ala-233was constructedwith the primer 5'-GCCGCTTTCCGTTG"C*CCCGAATTTCGT-3' without strandselection,transfecting the heteroduplexinto competentBMHTI-lSmutL with a lawn of BMH7l-18. MutantsLys * Ala-82,Arg----Ala-86,Lys - Ala-230, and Glu - Ala-235wereconstructedwith primers 5'-GCTTTTTG*C*CCCGCTC-3" 5'-TCAGCGTGG*C*CTCGCTTT-3" 5'-CCCGAATG*C*CGTGCCG-3" ANd 5'-GCCGCTTG*CCGTTT-3' in double priming with the strandselectionprimer SEL3 on Ml38l9TyrTS template. Heteroduplexwas transfectedinto competentHB2155 with of the TyrTS gene a lawn of HB215l. The codingsequence (Winter et al., 1983)wascheckedwith a family of sequencing primers(Wilkinsonet al., 1984). An asteriskfollowsa mismatchedbasein the mutagenicprimers. Purification of Enzymes. Mutant enzymeswere expressed in E. coli TG2 hostsfrecA form of TGl (Gibson,1984)]and purified to electrophoretichomogeneityby modificationof (Wilkinsonet al., 1983). An overnight existingprocedures cultureof E. coli TG2 in 2xTY medium(16 g of tryptone, 10 g of yeastextract,and 5 g of NaCl per liter) wasdiluted

FERSHT

ET AL.

100-foldinto 5 mL of fresh medium and grown to early log phase(Asso- 0.05) beforeinfectingwith recombinantM13 at high multiplicityof infection()100). Growth wascontinued at 3'7"C for t h beforebeing transferredto 500 mL of fresh, prewarmedmedium and shakenovernight. The cells were harvestedby centrifugationat 7000 rpm for l5 min, resuspendedin a buffer containing50 mM tris(hydroxymethyl)hydrochloride(Tris-HCl) (pH 7.8), 1 mM aminomethane ethyl enedi ami netetraacetiaci c d (E D TA ), 5 m M 2fluoride, and 0.1 mM phenylmethanesulfonyl mercaptoethanol, and immediatelylysedby sonication.The lysatewas heated enzymesfrom at 56 oC for 40 min to precipitateendogenous E. coli and clarified by centrifugationat 15000 rpm for 30 min. The protein precipitatingat J\Vo sattrating ammonium sul fate(0' C ) w asdi al yzedfor 1.5-3hat 4 oC aga inst50 mM potassiumphosphate(pH 6.5), 1 mM EDTA, 0.1 mM fluoride, and 0.1 mM tetrasodium phenylmethanesulfonyl tyrosyl adeny(to removeany enzyme-bound pyrophosphate late). After further dialysisfor 1.5-3 h againstthe samebuffer the proteinwasappliedto minustetrasodiumpyrophosphate, a 2 x 5 cm columncontainingDEAE-TrisacrylM equilibrated in the samebuffer. The columnwaswashedwith the starting buffer and elutedwith a gradientof 50-350 mM potassium phosphate(pH 6.5) containingI mM EDTA and 0.1 mM fluoride. The enzyme,which eluted phenylmethanesulfonyl at about 150mM salt,wasdialyzedagainst20 mM Tris-HCl, fluoride I mM EDTA, and 0.1 mM phenylmethanesulfonyl beforepurificationon a PharmaciaFPLC Mono Q column (Low eet al ., 1985). Kinetic Procedures All experimentswere performedat 25 oC in a standard buffer containing144 mM Tris-HCl (pH 7.78), 0.14 mM fluoride,and 10 mM MgClr. (ATP phenylmethanesulfonyl was addedas the magnesiumsalt to maintain the free Mg'* at 10mM.) Actiuation The rate constantsfor the formation of enzyme-boundtyrosyl adenylateby all the mutantsother than Glu - Ala-235weresufficientlylow that the reactioncould be monitoredby manualsamplingand trappingE'Tyr-AMP disks. A solutionof V4 mM Tris-Hcl-l0 on nitrocellulose mM MgCl2containing[taC]Tyr (210or 522 mCi/mmol) and (0.005unit/ml) of total yeastinorganicpyrophosphatase volume150pL wasincubatedat 25 "C; l5-30 p"Lof enzyme (2-10 pM) in the samebuffer was addedto initiate the reaction. Experimentsto determinethe valuesof Knafor ATP were generallyconductedat 40 pM tyrosineand those for determiningthe Ky for tyrosineat l0 mM ATP. Samples (20 pL) were periodicallywithdrawn and layeredonto a nidisk which wasundermild suction. The disk was trocellulose then washedwith 5 mL of Tris-HCl-MgClr buffer, dried in scintillant,and the underan infraredlamp, and suspended retainedE.ItaC]Tyr-AMP wasassayedby scintillationcounting. The reactionsfollowedfirst-orderkinetics,and seven sampleswere taken over five half-lives. The reactionswere followedto completion()10 half-lives)to obtain end points. Pyrophosphorolysis.A solutionof enzyme-bound[1oC]Tyr-AMP wasobtainedby incubatingenzymein a buffer at pH 6 [130 mM [bis(2-hydroxyethyl)amino]tris(hydroxy(Bis-Tris),10 mM MgCl2l in the presence methyl)methane of 25 pM [taC]Tyrand 25 mM Mg-ATP. The E'Tyr-AMP complexwasdesaltedon a SephadexG-50 (medium)column (l x 25 cm) with standardpH 7.78buffer. The enzyme-bound werestoredunderliquid nitrogen. The aminoacyladenylates weresufficientlylow that rate constantsfor pyrophosphorolysis the reactionscouldbe monitoredby manualsamplingas above.

TY.ROSYL-TRNA

SYNTHETASE

MECHANISM

voL.

27, NO. 5, 1988

1583

Table I: Activation of Tyrosine by Tyrosyl-tRNA Synthetaseo

K, (rrM)

frr (s-t) ktlK'^ (s-' 14-t; &-r (s-t) Koo (mM) k-tl Ko, (s-t Y-t; wild type t2 4.7 38 8080 16.6 0.61 27 200 -40b -2b Lys -* Ala-82 l3 50 730 Lys - Asn-82 l3 5.2 0.3 58 0.26 1.5 170 Arg - Ala-86 7.5 9.9 5 . 0x l 0 - 3 0.51 1.2 Arg - Gln-86 4 3.5 4 . 2x l0-3 1.2 3.5 x l0-3 2.5 1.4 Lys Ala-230 23 4.9 0.39 80 130 Lys - Asn-230 l9 3.4 2 . 6 x l0-2 7.6 l5 Lys - Ala-233 10.4 3.0 9.8 Lys - Asn-233 t2 0.2 0.38 oAll experiments conductcdin the standardbuffer at 25 oC and pH 7.78. Kr, the dissociâtion constantofthe E.Tyr complex,wasdeterminedby equilibrium dialysis. X'., the dissociation constânt of ATP from the E.ATP complex, k, and /ca, the rate constants for the forward and reverse reaction, and Ko, the dissociationconstant of PPi from the E Tyr-AMP.PP; complex, were determined from pre-steady-statekinetics. tK, for some mutantswasalsodeterminedkineticallyfrom the Éte of activationat subsaturating conconcentrations of ATP, and the valueswere found to be in excellentagreementwith thoselrom equilibriumdialysis. The blank entriesin th€ tableoccur wherethe valuerof (/â and KD are too high (>50 M and >.10mM, respectively) to be determined.rt3and *,3 are consequently inac.essible, bur the ratioskj/('! and i-r/KDe may iiill be determinedwith precrsron, enzyme

rK'" (mM)

The reaction was initiated by the addition of tetrasodium pyrophosphate to a solutionof 100-400nM E.[taC]Tyr-AMP in the standardbuffer (both solutionshaving beenincubated at 25 oC) to give a final volume of 130 pL. PyrophosphateExchangeKinelics. Thesewere performed for the mutant Glu * Ala-235 in the standardbuffer (Wilkinsonet al., 1983). Binding of Tyrosine. This was determinedby equilibrium dialysis(Fersht, 1975). Analysis of Kinetics. The first-orderrate constantsfor the hydrolysisof E.Tyr-AMP (kJ are comparablein somecases to those for its formation (k1, eq 3a). The observedrate kr

e

.;,

*;'

kh

E.Tyr-AMP + PPi --;

E * Tyr + AMP (3a)

constant for the formation of E.Tyr-AMP kobrdis given by kobsd= kr + kh (4) and the steady-stateconcentrationof E.Tyr-AMP ([E.TyrAMPlss) by (5) [E.Tyr-AMP]ss = lBlolh/(kr + ftr,)] where [E]e is the total concentrationof enzyme. [k6 is the sum of the rate constantsfor dissociationof the adenylateand direct hydrolysis(Wells et al., 1986).1[E]s may frequently be measuredby [E.Tyr-AMP]55 at saturatingconcentrations of substrates.Alternatively,the enzymewas assayedby its A2ssas we find that mutants are generallygreater than gOVo activeon activesite titration. Thus kr = kou.a[E.Tyr-AMP]ss/[E]o (6) For reactionswhich are too slow to follow to completion,the valueof /ô* i-:4,/

oa ,fo',, " \. Cvs-35- sH

His-48

Asp-l76-cq--' \. Tyr-34- on

,/o

Cys-35-sH

His-45

Thr-40

ïhr-51

His-48

E[Tyr-ATr1*

E.Tyr.ATP Arg-86.r, r.*"

\ï2

Y ffi-.;t.""

\ Fi "51 o."

'

,\o.t--"';',/.:7

ryrlos-o' Ho

L.{

t\. Asp-176-cÇ--TYr-34- oH

",i

Gln-l73-c(o-l---n. ---H-N-CCq-P-O

Asp-78-cd--'?-i:"

-- "(j

"'...

",i_rP3o

!ri.

- -^-'

His45

tn'fo

\rr,r,

.u=*r"'i;...

Arg-86a^ -NH

His-45

#

_à

\-ro"

lys-az---*"""...

-fo'::::

Grn-170-c(NH, -o-----n.*"

ET AL.

i){"--.r-;;t$t'

. Lys-f3O HrN.w * Lvs-233 HrNv/.