Affinity labelling of tryptophanyl-transfer RNA synthetase

J. Nol.

Biol.

(1977) 113, 475-501

Affinity Labelling of Tryptophanyl-Transfer VALERI

Institute

DMITRI

Z.

AKHVERDYAN,

LEV

L.

RNA Synthetase KrsSELEvt

of Molecular Biology of the USSR Academy of Sciences Vavilov Xtt.. 32, Moscow V-312. 117312 USSR G. KNORRE,

Institute

OL’GA

of Organic Academy

(Received

I. LA~RIK

AND

GEORGI

Chemistry, Siberiwn Branch of Sciences. Novosibirsk

7 October 1976, and

in revised

form

A. NEVINSKY

of the UXBR

7 March

1977)

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme. Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, q. An ATP analogue, r-(p-azidoanilide)ATP does not serve as a substrate for enzymatic aminoacylat’ion of tRNATrP but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K, = 1 x 10m3 M (K, for ATP = 2x 10m4 M). The covalent photoaddition of azido,4TP$ results in complete loss of enzymatic activity in both the ATP-[32P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme. been found from complex formation at Two binding sites for tRNA Trp have molecules bind, with Kdis = pH 5.8 in the presence of Mg z+. The two tRNA 3.6 x 10-s M and Kdis = 0.9 x lo- 6 M, respectively, pointing to a strong negative co-operativity between the binding sites for tHNA. IV-chlorambucilyl-tryptophanyl-tRNATrp and TRSase form a complex with of 10 mM-Mg2+. This value is h-es = 5.5 x 10ea M at pH 5.8 in the presence similar to the value of Kdls for tryptophanyl-tRNA of 4.8 x lo- * M. Under the same conditions a 1: 1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilylLTrp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of t.RNATrr whereas the tRNA devoid of tRNAT’r does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the t To whom correspondence should be addressed 2 Abbreviations used: azido-ATP, y-(p-azidoanilide)adenosine triphosphate; TRSase, tryptophanyl-tRNA synthetase (EC 6.1.1.2); R-Trp-tRNA, a residue of ambucilyl-tryptophanyltRNA covalently bound to the protein; Cl-R-Trp-tRNA, N-chlorambucilyl-tryptophanyl-tRNA; chlorambucil, y-p-bis(2-chloroethyl)-aminophenyl-butyric acid; -R-tryptophan, a residue of ambucilyl-tryptophan covalently bound to the protein: BBC, benzoylat,ed diethylaminoethyl cellulose. 475

Exhaustive alkylatiorr of TRSase l)artiall>itllriblts t,lrc rcwctir)Il of’ A’I’I’ ~3~P]pyropho”phat’e exctlaIlgc alrd conlplrtc3ly t)lorks t,tlcx ar~tirloac~~li~t~iot~ of tRNATr*. Cleavage of the t)RNA Mllicll is covalently hourld to TRSwse rcstorchs both the ATP--[“2P]pyrophosptlate c’x(‘l langcs and arninoacylation act,i\-itp. The TRSase whicll is cor2llently-bolulcl to R-Trp-tRNA is ablr> to ir~cwqx~~~~t~~ only one ATP molecule per dimoric enzyme into tile active cent,rcx. ‘I’llis cloubl~~ modified enzyme is completely enzymatically inact,i\,r,. .Kcmoval of tllr tKNA residue from the doubly modified enzyme: results it1 the formation of tt,c- doriv;ltive with one blocked ATP site. Tllerefore, a “single-site” TKSast~ may i)(b generated either b>- alkylation of t)lle cxllzyme \vith (‘I-R-Trp-tKNA or after ttrc, removal of covalently bound tKNA from the doubly labelled protc,itl. Tryptophanyl-tHNA synthetase containing blocked ATP and/or tKNA binding site(s) seems to bo a useful tool for investigation of 11~Lgative co-operativity anal may help in the elucidation of t,lrc, s;tructurP filrrctiotr rc~latiotlsllips betw(l(.ll the active centrfv.

1. Introduction Aminoacyl-tRNA synthetases (EC 6.1.1) playing the role of a vocabulary between the languages of nucleic and amino acids have three different substrates. Their structure and function have received wide attention in terms of nucleic acid-protein recognition and also in terms of the catalytic mechanism (see reviews by Kisselev $ Favorova (1974) and 5611 t Schimmel (1974)). The primary structures of these enzymes have not been established as yet although an hypothesis on a probable catalytic mechanism has been put forward by Krayevsky et al; (1973). One useful method for studying the active centres of the enzymes is affinity labelling. The method (Backer, 1967) is based on the use of substrate analogues containing a group capable of reacting with the functional groups of the enzyme. In the case of the synthetases, analogues of all three substrates have been proposed which are capable of producing covalent complexes with the enzyme. For amino acids there are methyl halo-ketone derivatives (Frolova et al., 1973; Silver & Laursen, 1974), for ATP there are photoactive y-ATP amides (Budker et aZ., 1974; Ankilova et al., 1975), and for tRNA there are derivatives containing an active group bound to the aminoacyl residue of aminoacyl-tRNA (Bruton & Hartley, 1970; Santi et al., 1973; Lavrik & Khutoryanskaya, 1974; Bartmann et al.? 1974a; Gorshkova et al., 1975), and derivatives containing a photoreactive azido group bound to 4-thiouridine in Escherichia coli tRNA (Budker et al.. 1974; Schwartz & Ofengand, 1974; Schwartz et al., 1975; Gorshkova et al., 1976). In all the aforementioned cases, the active analogues were used in separate experiments. However, sequential modification of one enzyme by several affinity reagents may open new opportunities for the analysis of structure-function relationships between active centres of oligomeric enzymes. In the present work, we have made use of the two analogues, of ATP and of aminoacyl-tRNA (their structures are shown on page 477) for the modification of beef pancreas tryptophanyl-tRNA synthetase. TRSaset (EC 6.1.1.2) is an enzyme with a molecular weight of 108,000 to 120,000, consisting of two identical subunits of molecular weight 54,000 to 60,000 (Favorova t See footnote

to p. 475.

XFFlNITY

LABELLING

OF

Trp-tHNh

O-

R Y-

tryptophon chlorambusm

477

SYSTHETASE

tRUA

s de cQa n y!

‘ryplophany

IRNA””

et ul.. 1971; Gras et al., 1972: Kisselev, 1972). A simple preparative method for TRSase isolation has been devised; it enables one to obtain in four days 100 to 150 mg of enzyme from 1 kg of pancreas (Favorova ef al., 1974), thus making it possible to use this enzyme for structural investigations. The kinetic mechanisms of ATP-[V]pyrophosphate exchange and of tRNA aminoacylation have been thoroughly studied by Knorre et al. (1974) and Kochkina ef al. (1976a.b).

2. Materials

and Methods

(a) Materials Tryptophanyl-tRNA synthetase from beef pancreas was isolated according to Favorova et al. (1974). An enzyme form having a subunit structure a2 and a molecular weight of 120,000 was taken for the experiments. One band of molecular weight 60,000 was visualized under denaturing conditions after gel olectrophoresis. Total yeast tRNA was received from the Special Design Office for Biologically Active Substances (Novosibirsk) and conr-(p-azidoanilide).ATP and r-(p-azidoanilide)-[3H]ATP wCre t.ained 0.57& tRNATrP. synthesized as described by Babkina et al. (1975). N-llydroxysuccinimide ester of chlorambucyl was obtained according to Grachev et al. (1972). ATP (sodium salt), L-tryptophan and z-phenylalanine were from Reanal. Cetyltrimethylammonium bromide (Cetavlon) was from Schuchardt, [14C]tryptophan (spec. act. 54 Ci/mol) from Amersham. [32P]Pyrophosphate and [3H]ATP were from Isotope. Nitrocellulose filters AUFS (pore-size 1.5 pm) and HUFS (pore-size 0.4 pm) were from Chemapol. Sephadex G50 fine and GlOO superfine were obtained from Pharmacia. Silufol plates were from Kavalier, Norit A from Serva. Benzoylated DEAE-cellulose was a gift from Dr A. Treboganov. All other reagents were of analytical grade. (b) ATP-[32P]33yrophosphate

exchange

reaction

The ATP-[32P]pyrophosphate exchange reaction was carried out in 1 ml at 37°C according to Favorova et al. (1974). The incubation mixture contained (m&I) : tryptophan, 0.02; ATP, 10; [32P]pyrophosphats, 3; MgCl,, 10; Tris*HCl (pH 7.5), 50; TRSase, 1 x 10e5 to 5 Y 1O-5; bovine serum albumin, 0.16 mg/ml. Equal portions were withdrawn after 0, 5, 10, 15 and 20 min. The reaction was stopped by 5 vol. 5 06 trichloroacetic acid containing I 96 activated charcoal (Norit A) and 2 x 10m3 M cold pyrophosphat,e. After vigorous shakign the mixture was passed through AUFS nitrocellulose discs and washed with 10 ml water, 3 times. The disks were dried and assayed in toluene in an SL-30 scintillation counter (Intertechnique). (c) Kin,etics

of tryptophan-t&VA

formation

The> incubation mixture contained (mM) : Tris.HCl (pH 7.5), 50; MgCl,, 5; NaCl, 20; ATP. 3x 1O-2; [14C]Trp, 1 x 1O-3 (spec. act. 52 Ci/mol): yeast tRNA, 1 x 10e3; TRSase, 0.8 Y 1Om5 to 5x 10m5; gelatine, 0.5 “6. Tot,al vol. 0.5 ml, incubation at 37°C; portions were

478

V.

removed at various time intervals room temperature the precipit,abes wadred 3 times with 10 ml water. This procedure is more convenient, gives a lower background value.

Z.

rlKHVERT)YAN

E2’

A I,.

and added to 1 ml of 1 ‘$/o Cetavlon. After 20 Init) at, were transferred to AUPS nitrocnlluloso filters and After drying tile discs wcn colmt~rtl as described above. ttlan t)trca one described by Muench & Borg (1966) and

(d) Preparation

of 114C]try~~tophanyl-tRi~‘A

Enriched Trp-tRNA was obtained by a modified version of the method of Maxwell et al. (1968). Total yeast tRNA was chromatographed on the BDC column in a 0.4 DI to I.0 m-NaCl gradient in the presence of 10 IBM-MgCl,. The tR,NA Trp fraction obtained after the first stage of purification was aminoacylated \vith [14C]Trp (52 Ci/mol) under the conditions described above with the only difference that the concm of ATP was 2 mM, that of TRSase O-8 x 10m7 I\I and gelatine was not added. The volume of the reaction mixture was 200 to 500 ml. After 5 min of incubation at 37”C, 0.1 of the vol. of cooled 2 M-sodium acetate buffer (pH 4.5) was added and the incubation mixture was placed on ice. For separation of [l%]Trp-tRNA from non-aminoaoylated tRNA the solution was passed through a BDC column under cooling in 0.05 M-sodium acetate buffer at pH 4.5 in the presence of 10 mivr-MgCl,. The low molecular weight substrates were eluted from the BDC column with 0.4 M-NaCl. Non-acylated tRNA was elnted with 0.5 to 0.8 M-NaCl. Trp-tRNA was eluted with 0.8 M-NaCl (1.0 M-NaCl $- 20% othanol) gradient. The isolated [14C]Trp-tRNA was concentrated on the DEAE-Sephadex A50 microcolumn. The concentrated Trp-tRNA was precipitated with ethanol. The precipitate was collected by centrifugation, dried in tho cold under vracuum over P,O,. The [14C]Trp-tRNA content in the preparation obtained was 30 to 50% of tile total tRNA content. For obtaining individual tRNATrr before separation of Trp-tRNA from non-acylated tRNA an additional chromatographic step was performed on a BDC column buffered with 0.05 MTriseHCl (pH 7.5) in a 0.6 M to 0.8 If-NaCl gradient in tho presence of 10 m&I-MgCl,. This stage offered an additional 3.fold enrichment of tRNATr*. Then tRNATr* was amino acylated and separated from the non-acylated t,RNA as described above. The Trp-tRNA obtained was 80 to lOOo,o pure. To prepare non-acylated tRNA Trp the preparation obtained was deacylated in Tris.HCl buffer at pH 8.5 for 2 h a,t 25°C. (e) N-chZorambuciZyl-[14C]tryptophunyl-tR~VA

synthesis

Cl-R-[14C]Trp-tRNA was obtained by means of the reaction between [14C]Trp-tRNA and the N-hydroxysuccinimide ester of chlorambucyl. The conditions of this reaction differed from those (Bochkareva et al., 1973) in the method used for obtaining N-chlorambucilylphenylalanyl-tRNA by tho fact that the reaction was carried out at pH 4.8 and not at 5.8, because at pH 5.8 during 24 11 of reaction at 20°C a significant deacylation of tryptophanyl-tRNA was observed. For obtaining Cl-R-Trp-tRNA we used Trp-tRNA of 25 to 35% purity. The Cl-R-Trp-tRNA obtained was precipitated by adding ethanol, and was reprecipitated twice with ethanol. The precipitate was collected by centrifugation, and kept cool in a vacuum desiccator over PzOa. For determination of N-acylation of [l%]Trp-tRNA, alkaline deacylation of the product of the reaction was carried out at pH 9 for 24 h at 37°C. [14C]Trp-tRNA was used as control. The hydrolysate was ohromatographed on Silufol plates in aqueous methanol. Less than 50/ of the total radioactivity had the mobility of tryptophan, whereas the rest formed a radioactive spot corresponding to N-acyltryptophan. The degree of N-acylation of Trp-tRNA found in this way was approx. 95%. (f) Preparation of labelled tRNATrp An equal vol. of NaIO, (80 mg/ml) was added to 0.2 ml of individual tRNAT’P (1576 After 15 min of incubation in the dark pmol/A,,, unit) containing 10Azeo units of tRNA. at 25”C, 0.05 ml of 2 ~-Kc1 was added and it was kopt on ice for 5 min. The precipitate was removed by centrifugation, and 0.05 ml glycerol was added to the supernatant containing tRNA with oxidized 3’.terminal ribose. After 15 min, tRNA was precipitated with ethanol, reprecipitated 3 times and finally dissolved in 0.5 ml of 0.1 M-Tris.HCl (pH 7*5), 0.2 pmol NaB3H4 was added to the tRNA solution and after 30 min incubation at 25°C the pH of the solution was adjusted to 4.5 with acetic acid and left for several

AFFINITY hours activity

for

LABELLING

the removal of the tRNA

of excess preparation

OF

tritium. After was measured

Trp-tRPiA a-fold

RYNTHETASE

479

reprecipitation, the specific radiofound to be equal to 10 cts/min per

and

prr101.

(6) I’hotoaddition

of azido-ATP

to TRSase

T11e pliotoaddition of azido-ATP to TRSase was carried out using the equipmriitj described (Bazhulina et al., 1974). Ultraviolet light irradiation was at 320 t’o 350 run and VC!. The reaction was run in 0.1 &r-Tris.HCl (pH 7.5) in tlic presence of 5 mM-MgCl,. The covalent attachment of azido-[3H]ATP was assayed hp radioactivity after protein adsorpt,ion on HUFS nitrocellulose discs. The non-bound azido-ATP was washed out with 0.05 M-Tris.HCl (pH 7.5) containing 10m4 nr-ATP. (II)

formation

Complex

between

TRSase

and

N-chlorambucilyl-Trp-tRXA

The complex formation between TRSase and Trp-tRNA or Cl-R-Trp-tRNA was carried out in buffer A: 0.025 M-sodium acetate (pH 5.8). 10 m&r-MgSO,, 1 X 1O-4 M-EDTA. The mixture contained 0.5 x 10e7 to 1.25 x lo-’ 3r-TRSase and 0.4 x 1Om8 to 1.7 x 10m6 31 [14C]Trp-tRNA or Cl-R-[r4C]Trp-tRNA. The volume of the incubation mixture was 0.1 ml. TRSase was the last reagent to be added. After 2 min incubation in the ice bath tho mixture was diluted with 1 ml of precooled buffer A and passed through the HUFS nitrocellulose filters prewashed with the same buffer. The procedure is similar to that of Yarus & Berg (1967). The filt,ers were washed 3 times with 3 ml of buffer A, dried and counted. In the experiments on the influence of low molecular weight substrates on the complex formation between TRSase and Cl-R-Trp-tRNA t’he concentrat’ion of the substrates was 1 ix 10-3 111. (i) Alkylation of TRSase by Cl-R-Trp-tRXA The alkylation of TRSase was TRSase and 1.2~ 10m6 to 2.5~ of alkylation wa,s estimated by cellulose filters under conditions of TRSase and Cl-R-Trp-tRNA. with I to 2 ml of 0.05 M-TriseHCl was passed through presoaked 5 ml of the same buffer. (j)

Isolation

performed in buffer A containing 1 x lo- 6 to 2.5 x 10m6 AI10V5 M-Cl-R-Trp-tRNA at 37°C for 4 to 7 h. The degree the amount of radioactivity in the protein fixed on nitroleading to the dissociation of the non-covalent complex 10 to 20.~1 portions of the reaction mixture were mixed (pH 7.5). After 5 min incubation at 20°C the solution HUFS filters, and the filters were washed 3 times with

of covalent

TRSase-R-Trp-tRXA

complex

Tlie covalent complex of TRSase and R-Trp-tRNA was isolated on a Sephadex Cl00 column under the conditions in which the non-covalent protein-nucleic acid complex dissociates: 0.4 M-NaCl in 0.05 M-Tris.HCl, pH 7.5 (buffer B). The portions of the reaction mixture were applied to the Sephadex GlOO column (0.5 cm x 40 cm) equilibrated wit,h buffer B. The elution was done with buffer B at 2O”C, flow-rate 6 ml/h. fraction vol. 0.3 ml. 0.1 -ml portions were removed for radioactivity measurements. The same incubation mixture at zero incubation time was used as a control, Parallel to the estimation of the degree of TRSase alkylation, 5-~1 portions of TRSase and Cl-R-Trp-tRNA mixture (experiment), and TRSase and tRNA mixture (control) were added to the reaction mixture for the determination of the tRNA aminoacylation rate and ATP-lz2P]pyrophosphate exchange. The ratio of the initial rates of each of these reactions in the experiment and the control was ~xxd to calculate t,he degree of enzylne inactirat’ion caused by affinity labelling. (k)

Modijcation

of

TRSase

by tw:o afinity

reagents

modification of TRSase with a&o-ATP and Cl-R-Trp-tRNA The mixture of TRSase (4.2 x 1O-6 M) and azido-[3H]ATP (1 X 10-4 M) in 0.1 M-Tris.HCl (pH 7.5) containing 10 mM-MgCl, was irradiated by U.V. light as described earlier. The 5-~1 portions were withdrawn before and after irradiation to determine the enzymatic activity in the aminoacylation reaction. To measure the degree of covalent attachment of azido-[3H]ATP to the enzyme as described above we removed 20-pl portions before (i)

Sequential

480

I’.

%. ;\KHVE:HD\‘.IN

E7’

.-I/,.

;tltrl itftflr irrdiatiorr. ‘I’KSaw LVHS sepitrittrtl fr0tn tltf. ~‘.xwss of itzifif).AT I’ t)y gfbl lilt I’+ Ii011 through a Sephadrx G50 (finf~) f:olu~i~r~ (0.8 cuff 40 cm), rfpilihr;ttfd witlt 0.05 w ‘l?ris~H(!l (pH 7.5). Tire chlt~iorl was tlo,lc: with tllcl sa~nc’ bul‘ff~r, flo\\.-rat(B I 2 lnl/lr. fractiorl vol. 0.8 ml. The comples formatior~ bc~t\~c~c~r~ azido-I”H]ATl’-modifietl f\r~ymcl arltl (‘1-H. ‘I’rp-tjMKA was performed at pH 5.8, as described ahovr. Alkylat ioll of tnodified TliSasc! \~as performed as described above. TRSaso irradiated wit,h itzido-[3H]ATP, irk t,tlr presencc~ of ATP (I X I Om3 IV) to prfhect t,tlfx onzyuw from Inoflificn,t~iotr at, thfl spfvific sitns. \va,s rf111 as a control.

(ii) Sequential

of TRh’aase

mod~jkation

with

Cl- R-Trp-tR,VA

and

azido-A

7’1’

Alkylation of TRSase (8.8 :x, 10 T nz) was pt~rformc~d with (‘I-R-Trp-tRNA under conditions providing complete alkylation of t Ire cnzymc. 20.~1 portiorls ~~errx removed tirlring thcl course of the reaction for determinatiorl of the dfagree of covalent att,achment of t,ho reagent to the> enzyme (see above). 5~1 portions we’re takrn simultaneot~sly t,o measltre the activity of TRSase in the il’rP-[“2P]pgropllosptlatf, cxxcllangc reaction. Tile modificat’ioll of t,he alkylated TKHase wit11 azitlo-[“H]ATP was carried orlt~ ill several ways. First, irradiation of TRSase--R-Trp-tRNA was carried alit iI1 the presence of azido-[3H]ATP (1 x 1O-4 M) immediately after thtl alkylation rract.ion. As a control tllo same mixture was taken but 1 x 1W3 M-ATP was added. Second, t,hcL TRSaseK-TrptRNA complex was treated with RNAase t,o split tlte tRNA moiety. followed by irradiation in the presence of 1 X I OV 4 &I-azido-[ “H]ATp. Tn both cases. 5-J portions of tllcl reaction mixture were withdrawn for determination of ATT’ [ 32P]pyropllospllate exchange. And finally, TRSase modified sequentiallywith Cl-R-Trp-tK,NA and azido-[3H]ATP was digested with pyrimidyl RNAase to remove the tRNA moiety. The enzyme activity in t,llc ATP- [32P]pyroplrosphate exchange reaction bvas recorded before and aftar the nnclcasc treatment. After prolonged incubation of the photochemically cross-linked TRSase azido-ATP complex with RNAasr, no release of azido-(3H]ATP was notic-cxtl. To remove the azido-ATP and other low molecular weigllt components. the modified enzyme was dialysed overnight in a cold room against 0.05 M-Tris .HCl (pH 7.5) with 3 buffer changes. After the dialysis, the enzyme was irradiated in tile prest’ncc’ of 1 x 10. 4 XIazido-[3H]ATP and the exchange reaction was assayed. The covalent attachment of azido-r3H]ATP tKNA, was measured after the removal of thr: mixtures were passed tllrongll tlitrocrllldnsr measin-cld.

to TKSase, premodified with Cl-K-Trpt,RNA moiety wit,h RNAase. The reaction filtclrs anti thrl adsorbed radioactivity was

3. Results (a) Reaction

of TRS’ase with azido-ilTP

It, has been shown previously that y-(p-azidoanilide)-ATP hibitor of the aminoacylation reaction catalysed by E. synthetase (Budker et al., 1974). Here the substrate and azido-ATP with respect to TRSase were studied, Azido-ATP aminoacylation reaction catalysed by TRSase (not shown). Figure 1 point to a competitive inhibition of ATP binding value for azido-ATP is I x 10m3 M, compared with a K, Since

the

azido-ATP

affinity might

of the serve

analogue as a reagent

is only for

slightly affinity

lower modification

is a competitive coli phenylalanyl-tRNA inhibitor properties is not a substrate in The curves plotted by azido-ATP. The for ATP of 2~ 1O-4

than

that of

of the

inof the in K, M.

substrate,

TRSase.

After irradiation of the TRSase-azido-ATP mixture by U.V. light, covalent attachment of the affinity reagent to the enzyme was observed (Table 1). The kinetics of the photoaddition of azido-ATP followed by inactivation of ATP-[32P]pyrophosphate exchange and aminoacylation reactions catalysed by TRSase a,re given in Figure 2. The inactivation curve corresponds t,o the photoaddition curve and approaches 95% for both reactions. The maximum level of covalent binding under the given

AFFIXITY

LABELLING

OF

Trp-tRN.1

481

SYNTHETASE

I 1 2 [Azldo

Fro. 1. The rate of tryptophanyl-tRNA of a&lo-ATP at various ATP concentrations 2x10 4.

-ATP]

(mM)

synthesis in t,hv absence (0) and in the presence ((3,) curve 3, (in M) : curve I, 1 / 10 5; ~~Itt’ve 2, 5 Y IO-“;

conditions corresponds to four moles of azido-ATP per mole of TRSase. Table 2 illustrates the levels of modification of TRSase depending on the concentrations of azido-ATP. As is obvious, the level of photoinduced cross-links increases with increasing azido-ATP concentration. Since this enzyme, as mentioned above, is composed of two subunits with identical molecular weight and contains two tryptophan binding sites and two tryptophenyladenylate binding sites (Dorizzi et al., 1971), the observed excess of azido-ATP molecules bound to TRSase molecule may be attributed to a non-specific reaction between the analogue and the enzyme, i.e. outside the active centre. As is shown separately, azido-ATP does react covalently w&h other proteins,

TABLE

Photochemical

reaction

Incubation

1

between TRXase

and azido-1 3H]ATP Radioactivity retained nit~rocellulose filters, (cts/min)

conditions

on

Controls TRSase + azido-ATP in dark Azido-ATP, irradiated without enzyme The same, irradiated 30 min Azido-ATP, irradiated 60 min without followed by addition of TRSase and additional irradiation 30 min

90 ruin

300 450 350

enzymes 650

Experiment TRSase TRSase 4.2~ 10e6 30 min if not specified. same activity in both ATP irradiated without

and azido-ATP,

irradiated

together

4800

M, azido-[3H]ATP 1 x 10e4 M. Incubation at 8°C in Blanks were not subtracted. TRSase irradiated without reactions as non-irradiated enzyme. The experimental enzyme for 30 min ~a.9 taken for all furt,her calculations.

0.4 ml, irradiation azido-ATP has the value minus azido-

482

Time of lrradlotlon

(mln)

FIG. 2. Covalent binding of azido-ATP to TRSase. (a) Kinetics of azido-[3H]ATP binding induced by irradiation. Concent,ration of TRSase, 0.4 mg/ml; azido-ATP, 5 x lo-* M. (b) Time-dependence of the activity of TRSase incubated with azido-ATP. ATP-[32P] pyrophosphate exchange (x ), tRNA aminoacylation reaction (0). The remaining activity was measured as a ratio of the initial rates catalysed by irradiated and non-irradiated enzyme. 5.~1 portions were removed from irradiated and non-irradiated samples, kept in the dark and added to 0.5 ml of the solution for determination of the catalytic activity.

TABLE

2

Dependence of the extent of photoinduced coupling of a&o-ATY on the concentration of the analogue Azido-ATP (M)

Ratio of photochemically coupled azido-ATP t,o TRSese (M/M)

1 x 10-S 1x10-4 1 x 10-S 6x10-3 Incubation

and TRSase

0.2 1.5 3.4 5.7 t,ime 30 min,

TRSase

0.2 mg/ml.

for example, bovine serum albumin. Therefore, to reduce the contribution of nonspecific TRSase modification, low concentrations of azido-ATP and enzyme were used for the subsequent experiments. The protective action of ATP against both photoaddition and photoinactivation was noticed when the reaction of azido-ATP and enzyme was carried out in the presence of ATP (Fig. 3). For instance, in the absence of ATP under the given con-

* n”T’TTmXT

6

LABELLING

OF

Trp-tKN.1

SYNTHETASE

(a)

i ~~~~~-j "'

t t

2-

-~~ ~

483

/'

6L

FIG. 3. Protective action of ATP against photochemical modification of TRSase. (a) Kinetics of [‘*C]tryptophanyl-tRNA formation in the absence of bound azido-ATP (0) and after photo-induced cross-linking (0). Photolysis time, 30 min; azido-ATP, 1 x lo-* M. (b) The same as (a) but 4 x 10m3 M-ATP was added to the TRSase-azido-ATP mixture. (c) The same as (a), 4 mu1 of a&lo-ATP were attached to 1 mol of TRSase (0). Control (0). (d) The same as (c), but in the presence of 4 x 10m3 wATP during irradiation. Before (0) and after (0) irradiation and covalent binding of 2 mol of azido-ATP per mol of TRSase. (e) Dependence of the degree of TRSase modification on t,he concn of ATP in the react,ion mixture. Azido-ATP, 1 x 10m4 M. Concentration of TRSaw in (a) and (b), 0.2 mg/ml; (c) and (d) 0.5 mg/ml; (e) 0.1 mg/ml.

ditions, 1.5 moles of azido-ATP were bound per mole of TRSase, accompanied by 600,/, inactivation of the aminoacylation reaction. In the presence of BTP, only 0.5 mole of azido-ATP was covalently joined to the enzyme whereas the enzyme was not inactivated at all. When four moles of azido-ATP were bound per mole of TRSase irradiated without ATP, complete inactivation of the aminoacylation reaction was observed (Fig. 3(c)). H owever, irradiation of the azido-ATP-TRSase mixture in excess of ATP leads to photoaddition of only two moles of azido-ATP per mole of TRSase. and again the enzyme was not inactivated in any way (Fig. 3(d)).

4s-L

T’.

%.

11 K HVERJ,>-.\S

157’

.1 L;.

Figure 3(e) shows the protjcxc+ ive action of Vi~riOUs c:oncc~ntl.Htioris of’ ATl’ against the photoaddition rt~actiorl. In thcl ah~ru~~ of ATI’. tjtrrclca rnolt>s of azido-ATI’ pi mole of TRSasr wcw c~ovalcntly hound. L\ft,(lr t.hv addit.ion ot’ .\‘I’t’ thcb protf~ction increased progressively \vith the incrcasct in ATP conccntrat ion. Stlvcsrt hclfw, complete protection was not’ achieved: and the protection curve rcbuchcd a plateau corresponding to one mole of azido-ATP per mole of TRSase. This rrsidual Icrel ()I’ photoaddition in t,he given conditions may be att’rihuted to a non-specific modification of the enzyme whereas the level of two molrs of azido-ATP per mole of TRSasr~ may be taken for affinity modification, The level of non-specific modification was successfully reduced to 0.4 mole/mole TRSase when the enzyme concentration was diminished fourfold as compared with the concentration used for the experitnrnt, described in Figure 3(c). Thus, azido-ATP is a competit,ive inhibitor \vith respect to ATP in the aminoacylation reaction. Also, at the covalent’ attachment site it causes irreversible inhibition of aminoacylation and ATP-[32P]pyrophosphate exchange. ATP prot’ects TRSase both against photoaddition of azido-ATP and irreversible inactivation. The data under discussion testify that the binding centres for azido-ATP and ATP are the same, and that under optimal conditions covalent addition of azide-ATP azido-ATP may hfa used for proceeds predominantly in these centrcs. Consequently. affinity labelling of the active centre of TRSase. (11) Reaction

of TRSase

with Cl-R-Trp-tRNA

To investigate affinity modification of TRSase wit’11 a Trp-tRNA analogue one needs information concerning the number of tRNA binding sites per enzyme tnolecule. Figure 4(a) shows the dependence of complex formation between TRSase and Trp-tRNA on the Trp-t,RNA concentration at pH 5%. Proceeding from the effectiveness of the absorption of the complex on nitrocellulosc filters we have found that maximum binding of TRSase and Trp-tRNA corresponds to one mole of Trp-tRNA per mole of TRSase. Similarly it, was demonstrated (Fig. 4(b)) that the stoichiometry of the complex formation between Cl-R-Trp-tRh’A and TRSase corresponded to one mole of reagent per mole of enzyme. Figure 4(c) summarizes the results on the competit’ion of tRNATrP and Cl-R-Trpfrom the complex tRNA for TRSase. Transfer RNA Try displaces Cl-R-Trp-tRNA with TRSase whereas the t,otal tRNA, without tRNATrP: does not influence the effectiveness of the complex formation. Using the data presented in Figure 4(a) and (b) the Kdis values for Trp-tRNA and Cl-R-Trp-tRn’A were calculated (Fig. 4(d)). These values are equal to 4.8 x lo- * M and 5.5 x 1W8 M for Trp-tRNA and Cl-R-Trpt.RNA, respectively. This means t,hat t,he affinit,y of TRSase for Trp-tRNA and its analogue is the same. In Figure 4(d) the curves intersect t)he ordinate at 1 indicating the ability of TRSase to bind only one mole of Trp-tRNA and its analogue. Summarizing the data presented in Figure 4 we may conclude that Trp-tRNA and Cl-R-TrptRNA behave similarly during the complex formation with TRSase, i.e. the analogue forms a specific enzyme-substrat,e complex with TRSase. The complex formation between TRSase and uncharged tRNA (depending on the concentration of the latter) is shown in Figure 5(a). The curve is distinctly biphasic: the initial part corresponds to the 1: 1 stoichiometry whereas the binding of the second tRNA molecule to an enzyme molecule requires a much greater excess of

2

2 1

i

I-

.

1.

486

[3H]tRNA

(pMi iai

r------

-----ll_‘--

1

FIG. 5. Complex formation between TRSase (l-7 x 1W7 $1) and [%]tRNAT” (a); determination of K, for tRNATrp (b) ; and Scabhard plot fur &termination of the tRNA-binding sibes (c).

z\F”FINITY

LAHELLING

OF

‘I‘rp-IRS.\

SYSTHETASE

487

tRNATrP with respect to TRSase. At the highest tRXiA concent-ration the stoichiomet,ry of the complex was 1 : 1.9. The dependence of IEJ/jE .S] nerms l/[Sj is also biphasic. Kdis for the first tRNA molecule is 4 x lo- 8RI.and for the second,1 x 10-6~~. The data are presented as a Scatchard plot in Pigurt~ 5. According to Figure 5 Kd,, for the first tRiYA molecule is 3-6 s 10- R M. and for thra second 0.9 x 10- 6 xr. Since beef pancrease TRSase is composed of two probably identical subunits (Fa~orova ef /cl.. 1971,1974; Gros et nl., 1972: Kiss&v. 1972) it is most probablr Ohat the observed 25fold difference in t,hc afliniby is a consequencr~of negative co-opcrativity between tRNA binding sites. The negative co-operat,ivity for bryptophanyl-tRNA and its analogue. CI-R-Trp-tRNA. is expressed more st,ronglg than t,hat for tRIY’AT’p binding.

T

Time

(hl

Froct~on

no

2

(a) ditions (b) NaCI, (---peak (b) (c) .>. ,>J

FIG:. 6. Alkylation of TRSase (1 x IO-’ M) with C1-R-[.14C]Trp-tRN.~ (5 x 10~’ M). 1)etermination of degree of alkylation with nitrocellulose filter technique under the conprovoking dissociation of the non-covalent enzyme--tRKA complex. and (c) Gel filtration through Sephrtdex Cl00 column (0.8 cm j, 40 rm) at, pH 7.5 in 0.4 nzof t,he TRSase and Cl-R-Trp-tRNA mixture. ---), A260”m; (. . . . . .), radioa&ivity. Peak 1, TRSasrx or ~~nzymn-R-Trp-iRNA; 1, C1-R-[3H]Trp-tRNA. Gel filt~ration immediately after mixing TRSaso and C’I-R-~.3H]Trl~-t~RN;\. Gel filtration after incubation of the TItSase-C1-R-[3H]Tr1)-tRNA mixture for 5 h at 37°C.

488

V . Z . X I< H \r E R I) Y .1 N X 7

.-l I,.

The kinetic curve in Figure 6(a) shows the alkylation of TRSasc with Cl-R-‘l’rptRNA. The covalent nature of the binding of Cl-R-Trp-tRNA to TRSase is prov~~d by the gel filtration experiment (Fig. 6(b) and (c)) in which the reaction mixture of TRSase and Cl-R-Trp-tRNA was passed through the column under the conditions in which the non-covalent complex was unstable. When the isolated TRSase-R-Trp-tRNA complex (B’ig. 6(c), peak 1) was passed through a nitrocellulose filter as described in Materials and Methods more than 90% of the radioactivity was retained by the filter. If a non-incubated mixture (Fig. 6(b)) was passed through the filter under the same conditions less than 50/, of the radioactivity was retained as compared with the previous case. We conclude, therefore, that a good correlation exists between gel-filtration and nitrocellulose filter techniques and the latter can be used for quantitative measurementas. The formation of the covalent bond between Cl-R-Trp-tRNA and the enzyme was also revealed after the tRNA residue was cleaved by pancreatic RNAase hydrolysis or by alkaline hydrolysis of the ester bond between R-Trp and tRNA. The results are given in Ta,ble 3. Figure 7 shows the dependence of the degree of alkylation of TRSase on the Cl-R-Trp-tRNA concentration. With the elevation of Cl-R-Trp-tRNA concentration of the the degree of alkylation increases, fivefold excess leads to the formation TABLE Stability

of the cross-link

between TRSase hydrolysis

RNAase hydrolysis

0

750 780

2

4

Cl-R-Trp-1RNA

FIG. 37°C.

7. Alkylation

of TRSase

and R-[14C]Trp-tRNA

against

Protein-bound radioactivity, (cts/min per pg) Before After hydrolysis

Type of tRN.A split&g

Pyrimidyl Alkaline

3

at various

6 lmol)/TRSose

enzyme/Cl-R-Trp-tRNA

730 740

8

IO (mol)

ratios.

Incubation

time

6 h,

AFFINITY

LABELLING

OF

Trp-tRNA

SYNTHET$SE

4x9

covalent enzyme-R-Trp-tRNA complex with t’he stoichiometry of one mole of reagent per mole of enzyme. Further increase of t,he Cl-R-Trp-tRNA:TRSase ratio does not affect the level of TRSase modification. Addition of sufficient tRNATrp to the reaction mixture for almost complete displacement of Cl-R-Trp-tRNA from the non-covalent complex with TRSase leads to the entire inhibition of the alkylation reaction. Total tRNA at the same concentration (without tRNATrP) does not inhibit the alkylation reaction of TRSase (not shown). The effect of alkylat,ion on the enzyme act)ivit,y in t’he aminoacylation reaction an d in ATP-[32P]pyrophosphatt: exchange is shown in Figure 8. Under the same conditions the kinetic curve of the covalent attachment of Cl-R-Trp-tRNA to TRSase reaches the plateau level of one mole of R-Trp-tRNA per mole of TRSase (Fig. 6(a)). Parallel to the increase in the degree of alkylat,ion of the enzyme t’he degree of irreversible inhibition goes up both for the aminoacylation of tRNA and for thr BTP-pyrophosphate exchange. However, the aminoacylation reaction is completely inhibited at the maximum degree of alkylation whereas bhe complete inhibition of ATP-pyrophosphate exchange: was not achieved. ;md the maximum inhibition was approximately 60 t,o 700,;, (Fig. 8).

1 0

I o-2

I

R-Trp-tRNA Fra. 8. ATP-[32P]pyrophosphate complete alkylation. TRSase, 4.2 x 10-s M; Cl-R-Trp-tRNA,

exchange

I 0.4

I

I 0.6

1

I 0.0

(mol)/TRSase ( x ) and

2.5 x 10e5

tRNA NI. Incubation

t I.0

(mol) aminoacylation

(0)

time

5 h, 37°C.

reactions

after

Summarizing the evidence of the ability of Cl-R-Trp-tRNA to be used for affinity labelling of TRSase it is reasonable to conclude that the given reagent meets all the requirements for an affinity reagent (Singer, 1967 ; Ankilova et al., 1975). Formation of the covalent bond between R-Trp-tRNA and TRSase may cause the loss of enzymatic activity due either to damage to the catalytic or substrate binding sites of the enzyme after the alkylation reaction, or to the shielding of the enzyme by the bulky tRNA molecule. This latter suggestion was proved by the fact that after removal of the tRNA by RNAase or hydrolysis more than SOo/O of the enzymatic activity was restored (Fig. 9). In spite of the tRNA splitting, the radioactivity loca’ted in the tryptophan moiety

490

f.

%.

.\KHVERl)Y,-\;lr

E’f’

,I 7,.

should remain lwund to the protck if the covalrnt bond hrtn-wn tt-Trp and thtb twzyme is stalk under the given conditions . As sew from Table 3 the radioac$ivity, i.e. R-1 14CITrp, was found hound to TKSase after the removal of tRN.4. The dat’a obtained show unequivocally that the covalent, bond het’wtaen the enzyme and R-Trp-tRNA is formed outside the act’ive centre and, moreover, the presence of the covalently atta,ched R-Trp: or R-7’rp-adt:nosinf, does not intwfcw with the catalytic a&ion or suhst’rate binding.

(b)

I

I 5

I IO Tune

I 15

(mm)

FIG. 9. Catalytic activity of the alkylated TRSase after removal of the tRNA moiety. The enzyme contained 1 mol R-Trp-tRNA/mol TRSase, was inactive in the aminoacylation reaction and 40% active in ATP-[32P]pyrophosphate exchange. Removal of tRNA was achieved (c). (a) Aminoacylation; (b), hydrolytically at pH 7.5 t-o 8.5 ((a) and (b)) or with ribonuclease (c) ATP-[32P]pyrophosphate exchange. Enzyme activity before modification (a), after complete modification with Cl-R-Trp-tRNA (0) and after splitting of tRNA ( x ).

(c) Influence

oj’ the low molecular

weight substrates on the alkylation

reaction

It has been shown earlier (Gorshkova & Lavrik, 1975; Gorshkova et al., 1976) that the kinetics of affinity labelling of E. coli phenylalanyl-tRNA synthetase with active tRNA derivatives are very sensitive to low molecular weight ligands. The changes in the rate of affinity labelling observed in the presence of ligands may be of specific and non-specific nature. The non-specific inhibition of the alkylation reaction might result

AFFINITY

LABELLING

OF

Tq-tRK;A

SYSTHETARE

491

from the reduction of the concentration of t,he active analogue due to its interaction N-chlorambucilyl-phenylalanyl-tRXA does not with added ligand. However. wlkylate ATP in solution or in the enzyme-substrate complex (Gorshkova & Lavrik, 1975). Cl-R-Trp-tRNA does not react with ATP and tryptophan after six hours incubation at 37°C as revealed after gel filtration of t,he reaction mixture containing [ 3H JATP or [3H]tryptophan and Cl-R-Trp-tRNA (not shown). Thus, the influence of' t'hr ligands on the alkylation ratBe of TRSase via direct’ chemical modification of Cl-R-Trp-tRSA may be ruled out). Figure 10 shows the inhibition of the alkylation reaction caused by ATP and amino acid. &\TP prevents the alkylation reaction, and the specificity of this effect is manifested by the observation that neither GTP nor CTP has any influence on the alkylnt,ion rat,fb. The protective action of the ATP increases progressively with the concentration and at, 5 rnnf-ATP the alkylation of TRSaw is completely inhibited. r,-Tryptophan also possesses protective activity. How-ever. the protection does not exceed 5Oqb even at 2 ;< 10e4 hl-tryptophan, which is two orders of magnitude higher than t,he K, (Fig. 10(b)). Presumably the protective action of trpptophan is not

FIG. 10. Influence of ATP (a) and Trp (b) on the alkyletion rate of TRSase. Concent,rations used (M): TRSaxe, 4.2~ 10-s; Cl-R-Trp-tRNA, 8x 10-s. (a) ATP, (0) (,) 3~10-~; (A) 6x10-*; (A) 5~10-~. (b) Trp, (0) 0; (Cl) 2~10-~; (A) 8x10-s; 2 x lW4. Phrnylalanine (1 x 1O-3 M) was used as a control ( * ). (c) Mixt~urr of 6 X 10e4 M-ATP 5 I 10~ B M-Trp ( n ); (a) control (without ATE’ and amino acid).

0; (n) -t

492

I-.

Z.

AliHVERtr,Y~\X

l!:‘l’

‘If,.

attributed to its hydrophobic nature alone since phenylnlanine has no effect, on thcb alkylnt’ion reaction. When both substrates are present the protective effect is additive (Fig. 10(u)). Since the reaction is run in the presence of Mg 2 + the aminoacvladenylate-enzyme complex may be formed under t,hese conditions. Hence, t,he increase in the protection might be connected only with the occupation of both binding sites. or with t’ryptophanyladenylate formation having higher affinity for the enzyme than any of the substrates taken separately. The interaction of the affinity reagent with the enzyme may be described by thr following equation : E+I+ErltE,,

1

where E stands for TRSase, I for Cl-Trp-tRNA and E, for alkylated TRSase. The first stage is complex formation between the enzyme and the inhibitor, the second one is the formation of the covalent bond between the enzyme and the reagent in the complex. The second stage may be considered as an irreversible step since within the course of the alkylation reaction no splitting of R-Trp-tRNA was noticed. To ascertain which of the stages is influenced by ATP and tryptophan the complex formation between TRSase and Cl-R-Trp-tRNA was performed in the presence of the ligands. It turned out (results not shown) that all the curves almost coincide and reach the plateau level of one mole of Cl-R-Trp-tRNA per mole of TRSase which is in agreement with the data shown earlier (Fig. 4). Therefore, the complex formation between TRSase and Cl-R-Trp-tRNA is not affected by the low molecular weight ligands. For certain aminoacyl-tRNA synthetases similar results were obtained where ATP and amino acids do not modify the affinity of the enzyme for tRNA or aminoacyltRNA (Yarus & Berg, 1969; Bartmann et al., 1974b; Evans & Nazario, 1974). Consequently, the protective effect against alkylation is attributed to the second stage. namely, to the chemical reaction between Cl-R-Trp-tRNA and TRSase. (d) Modi@xztion

of TRSase

by two afinity

reagents

Since it has been demonstrated that azido-ATP and Cl-R-Trp-tRNA are afinity reagents for TRSase this observation opens new paths for the investigation of the interaction between active centres via double modification by two affinity reagents acting sequentially. (i) ModiJication

of TRSase

with &do-ATP

followed

by Cl-R-Trp-tRNA

treatment

To exclude the influence of free azido-ATP on the reaction with the second affinity reagent the reaction mixture containing the enzyme and azido-ATP was passed, after irradiation, through a Sephadex G50 (fine) column. The enzyme modified by azido-ATP is localized in the excluded volume. The fractions were combined and the preparation was used for modification with Cl-R-Trp-tRNA (see section (ii), below). Initially we investigated the ability of TRSase modified with azido-ATP to form a non-covalent complex with Cl-R-Trp-tRNA. As is obvious from Figure 11(a) the modified TRSase is able to form a complex with Cl-R-Trp-tRNA. Moreover, the stoichiometry of E: tRNA is equal to unity, in other words it does not differ from the non-modified enzyme (Fig. 4).

AFFINITY

LABELLING

OP

Trp-t,RNh

4!El

SYSTHETASE

Time(h)

FJ~. Il. Complex formation between Cl-R-Trp-tRNA and TRSase premodified with azido-ATl’. (a) Formation of non-covalent complex under the conditions of stability for enzyme-tRNA complex (pH 5.8, 0°C). Modified (0) and non-modified (0) TRSase (1.25 x 10y7 M). (b) Alkylation of TRSase (0.5 x 10mg M) containing covalently bound azido-ATP. (O), TRSase treated with azido-ATP in the presence of ATP (non-specific modification, 2 mol/mol E) ; (a), TRSase, containing 4 mol azido-ATP/mol E (2 mol at active sites and 2 non-specifically, cf. Fig. 4(c) and (d)). Cl-R-Trp-tRNA, 2 x 1O-6 M.

The results of the alkylation of TRSase premodified with azido-ATP are given in Figure 11(b). It is clear that non-specific modification of the enzyme with azido-ATP does not prevent the alkylation reaction whereas a complete inhibition of the TRSase alkylation reaction with Cl-R-Trp-tRNA is observed if both ATP binding sites are previously blocked with azido-ATP. All these dat’a are in good agreement with the previous experiments on t’he influence of ATP and tryptophan on the alkylation of TRSase. It is impossible to label the enzyme with both the analogues if azido-ATP blocks the ATP-binding centres before alkylation takes place. This was the reason why we inverted the order of the modification reactions in the next section. (ii) Mod@cation

of TRSase

with

Cl-R-Trp-tRNA

followed

by azido-ATP

treatment

Two enzymatic forms obtained after alkylation of TRSase were treated with azido-ATP: (1) covalent TRSase-R-Trp-tRNA complex. In this case the photoaddition reaction was run both in the presence and in the absence of ATP. (2) Covalent TRSase-R-Trp-tRNA complex after splitting of the tRNA moiety. Non-modified TRSase in the presence of tRNATrP (the concentration of tRNATrp was the same as that of Cl-R-Trp-tRNA) treated with azido-ATP served as a control. The activity of all enzymatic forms was measured in the ATP-[32P]pyrophosphate exchange reaction before and after the photoaddition of azido-ATP (Fig. 12).

I 0

I

I

I

5

IO

15

Time

FIG. 12. ATP-[32P]pyrophosphate exchange TRSase, prealkylated with Cl-R-Trp-tRNA. Curve 1, Non-modified TRSese ; curve 2, alkylated sequentially; curve 4, the same as in 3, but after after the repeated treatment with azido-ATP.

The non-modified enzyme was mole of TRSase. The same amount of tRNATfp (Table 4). Approximately covalently attached to the enzyme analogue. Therefore, the level of under these conditions is equal to

modi$cution

same, 1 x 1O-2

in the x-ATP

Photolysis

time

+ tRSATrP

30 min,

1 x lOV4

photoaddit,ion

TRSase ; curve tRNA cleavage;

3, alkylat,ed curve 5, the

of

azido-ATP

to

and photoreacted same as in 4, but

4

TRXase

by azido-ATP

hzido-ATP, cross-linked with TRSaso (mol/mol)

subjected reaction

1.4 presence

of (1.5

TRSaseeR-Trp-adenosine TRSase

after

conditions

TRSase-R-Trp-tRNA The

activity

of the alkylated oarious

Enzyme preparation to photochemical

(mlnl

covalently bound to 2.6 moles of azido-ATP per of azido-ATP was covalently bound in the presence 0.5 mole of azido-ATP per mole TRSase was in the presence of excess ATP with respect to the specific modification of TRSase with azido-ATP two moles of the reagent per mole of the enzyme. TABLE

Photochemical

/

2.5 (cont,rol) M azido-ATP.

2.6

under

,\FFINIT\-

LAHP:LI,ISG

OF

Trp-tRK.1

SY?r”l’HET.1SE

496

Thew data correspond to the previous results on TRSase modification with azidoATP (Fig. 3(c) to (e)). TRSase cova’lently attachrd to R-Trp-tRKA (in the ratio of I mole of the reagent per mole of the enzyme) binds 14 moles of azido-ATP per mole of the enzyme under irradiation, i.e. only one binding centre out of two on a TRSase molecule is modified, taking into account 0.5 mole of non-specific modification. This is accompanied by complete inactivation of the c,xchangc activity retained after alkylation of TRSase with Cl-R-Trp-tRKA. When the tRPiA residue is eliminated from the complex hy hydrolysis with RSAastl. 2.5 moles of azido-ATP per mole of enzyme arc attached to the latter, i.e. this is equal t,o the amount) which is attached to the non-modified enzyme. In other words, tRNA cleavage results in covalent binding of one more mole of azido-ATP per molt of enzyme. Consequently, -R-tryptopha,u retaining its covalent bond wit,h TRSasc does not suppress eit’hcr t’he catalytic funct’ions of TRSase or the covalent binding of azido-ATP to TRSase. Thus. R-Trp-tRSA covalmtly bound to TRSaw (1 mole of reagent per mole enzyme) p&r&s one mole of azido-ATP per mole of enz>-me against the photoaddition reaction. Separately nrit$her -R-Trp nor tsRPiLATrP posscasrs any proCective a&on. After rxhaust’ive alkylation of TRSase with R-Trp-tRKA AOO,, of the activit)y is lost ill the ATI’-1 32P]p,vrophosphate exchange rcwtion (Figs 8 and 12. curve 2).

2

a Ll Y lli

6

: 4 t

Time

(mln)

FIG. 13. Aminoacylation rates of TRSaw after modification. Curve 1, Non-modified enzyme (control), E ; curve 2, after alkylation (1 mol R-Trp-tRNA/mol of TRSaae), E-R-Trp-tRNA; cwvc 3, after photochemical modification of the alkylated enzyme, E/R-Trp-tRNA; \azido-ATP F,‘R-Trp “\ ,azitlo-ATl’.

curve ’ 4 , aft,er

alkaline

hydrolvqis, ’

of

the

doubly

modified

enzyme ”

406

1’.

2.

~~KHVERI)YAN

1<7’ .-lL.

After subsequent modification of the alkylated enzyme with azido-A’I’L’ the remaining activity of the enzyme in the exchange react,ion is completely lost, (Fig. 1”. curve 3). However, aft’er the removal of the tRN14 moiety from the covalent complex 450/ of the activity is restored (Fig. 12. curve 4), i.e. t’hat part, of t,he nctivit,y which was previously inhibited aft.er alkylat,ion of TRSasc. If this partially react,ivated enzyme is subjected to another moditication with azido-ATE’ a complete loss of enzymatic activity is noticed (Fig. 12. curve 5). Bfter tRN,4 splitting from a, doublemodified enzyme the acylating activity is restored (Fig. 13) to the level corresponding approximately to the activity of the exchange reaction. This means t’hat the enzyme is able to perform the transaminoacylation reaction by a single subunit associated with an inactive one.

0.3

(a i

Fra. 14. Gel chromatography of the modified TRSase. (a) Position of the markers (bovine serum albumin, M, 67,000; native TRSase, M, 120,000). First peak TRSese, enzyme activity and absorbance; second peek bovine serum albumin, absorbance. (b) TRSase alkyleted with R-[‘%]Trp-tRNA (1: 1 molar ratio). First radioactive peak TRSase-R-[14C]Trp-tRNA; second peak free Cl-R-[‘W]Trp-tRNA. (c) TRSase after photoaddition of azidoj3H]ATP (3.5 mol/mol E). First radioactive peak TRSase-azido-[3H]ATP; second peak free azido-[3H]ATP. ) Protein concn (mg/ml) ; (. . .) radioactivity. ( Sephadex GlOO superfine column (0.8 cm x 40 cm) equilibrated with 50 m&r-Tris.HCl (pH 7.5) containing 0.4 M-N&I. Flow-rate 1.5 ml/h; fraction vol. 0.5 ml; void vol. ~4 ml. Protein concn in the presence of nucleic acid of ATP was measured as described by Ehresmann et nl. (1973).

AFFINITY

LABELLING

OF Trp-tRNA

497

SYXTHETASE

Chemical modification of a protein may cause a change in its oligomeric state, for example it may provoke its dissociation. Since TRSase which has been used in this study has an CI~structure we examined its molecular weight after the photo-addit*ion and alkylation reactions (Fig. 14). After gel filtration of the azido-&4TP-modified enzyme no protein was found in the elution volume corresponding to a monomeric molecular weight, of 60.000. In case of E-R-Trp-tRNA the monomeric form has a molecular weight of less than the native enzymt’ whereas the dimer is heavier. Figure 14 shows that the displacement of the peak towards a higher molecular weight takcxs plac~c and hence the dissociation doeti not occur.

4. Discussion When selecting substrate analogues containing should try to follow three main requirements. (1) The analogue must retain the properties affinity should remain sufficiently high despite subst#ra,te.

chemically

active

groups

of a competitive inhibitor some structural difference

one

and its from the

(2) The chemically reactive grouping must not possess too-high chemical selectivity. If the a&ive group is too specific it might happen that it would not find an appropriatth acceptor close to its location in the enzyme-analogue complex and, hence. the covalent binding of the substrate analogue would become impossible. Therefore we have given preference to the analogues containing chloroet,hylamino groups and even the less specific azido group (Patai, 1971). (3) The reactivity of the active group must not be too low. If this happens the yield of the product’ might be greatly reduced, and the duration of the reaction might be too long. ,4t the same time the reactivity of the active group must not be too high since in this case a non-specific modification may occur. In this respect Cl-R-Trp-tRNA, retaining the same affinity for the enzyme as the natural substrat,e Trp-tRNA and producing no non-specific binding outside the tRNA binding site, is a very good affinity reagent indeed. The close similarity of K,, values for Cl-R-Trp-tRNA and Trp-tRNA indicates that’ the amino group of the aminoacyl moiety in tryptophanyl-tRNA and probably the amino acid residue as a whole do not contribute to the interaction between the enzyme and aminoacyl-tRNA. The absence of any influence of the bulky substituent. group on the affinity between the tRNA analogue and TRSase shows that the aminoacyl group is located at the surface of the prot,ein molecule and probably has no strictly fixed site. Some other data (see review Kisselev & Favorova, 1974) fit this assumption. The long spacer between the active chlorine atom and the tRNA moiety of the affinity reagent allows the formation of a covalent bond far enough from the tRNA binding site. In fact, an almost complete rest.oration of the enzymatic function after splitting of tRNA proves clearly that the group in the protein molecule (i.e. any nucleophilic residue) which undergoes alkylation plays no role in enzymatic activity. In other studies with chemically active N-substituted aminoacyl-tRNA analogues (Bruton & Hartley, 1970; Santi et al., 1973; Lavrik $ Khutoryanskaya, 1974; Bartmann et al.. 1974aJ) the enzyme activity was also lost. However t,he reason for that, \vas notj clear since attempts to cleave tRNA w(bre not undertaken. Since in these

1RX

I'. Z. .\I
/CT :I/..

cixprriments the distance bct~wrrn t,tre active group and the A-C’% end of the tKS.\ molecule ~vas sllorter than in CLR-‘1’tpt~KSA thtl relation of tllca ;Imino ac*icl hid<& groups involved in cross-linking to (anzymat’ic function or binding remained ot)s;c~urr~. Azido-ATP is not an ideal affinity rc,agent since along wit,h specific modification of the ATP-binding site somr non-specific modification t,akes place. Since t~he affinity of ATP or TRSase is relatively low (Favorova et al.. 197-C) and is reduced furthc~r for the ATP analogue. it is necessary to us? sufficiently high concentrations of i\‘l’I’ analogue which in turn, due t,o high reaction ability, lead to the modification locatc~tl outside the active centre. In spite of these short,comings. azido-ATP may bc used as an nffinitp reagent in the case of TRSase as is shown in this work. In the case of phenylalanrl-t,R~A synthetase from E. coli this reagent’ does not produce specific covalently bound complexes and does not inactivate the enzyme although the photochemical addition does proceed effectively (Ankilova it al.. 1975). The observed difference bebween these two enzymes when treated with the same reagent is attributed to different properties of their ATP binding sites. In the case of TRSase the level of specific moditication remains cquaI t)o two rnolcls per mole of the enzyme, irrespective of the level of non-specific moditication. This saturat’ed lvith t,he analogue means that both ATP binding sites of TRSase ma; be and react with it. A big difference was observed in the behaviour of CI-R-Trp-tRNA as compared with azido-ATP. Firstly, at variance with azido-ATP the level of alkylation does not) depend on the concentration of (X-R-Trp-tRKA and is specific for the Trp-tRXA binding site(s). Secondly, in spite of t,he cc2structure and specific binding of t,wo moles of azido-ATP per enzyme molecule. no more t’han one R-Trp-tRNA molecule \\xz; non-covalently or covalently bound to TRSase wit,hin the limits of the tRh’A concentrations used in the experiments. The maximum stoichiomet,ry does not exceed 1.1 : 1 and the maximum possible level of alkylation was also equal to unity. Although. as was shown earlier, TRSase has two tRNA binding sites, t’he attachment of one R-Trp-tRNA molecule to the dimer is accompanied by inhibit’ion of t’he binding of the second subunit to the tRNA analogue, i.e. s strong negat’ivo co-operativitg and half-of-the-sites reactivity exist. The negative co-operativity shown here for TRSase in binding studies manifests itself also in kinetic experiments (Malygin et al.. 1976). The existence of two tR&A*‘* binding sites with unequal affinity was shown vcr!, recently for E. coli tryptophanyl-tRKA synthet’ase by equilibrium dialysis and sucrose densit,y gradient cent’rifugation (Muench. 1976). The data are in accord with those shown here for homologous enzyme from beef pancreas. Therefore TRSase joins the group of synthetases from various species which bind et aZ., 1975: Fersht. 1975: tRNA antico-operatively (Pingoud et al.. 1975: Bart,mann Persht et al., 1975; Jakes & Persht, 1975; Blanquet et al.. 1976). The stepwise modificat,ion by two affinity reagents performed for the first time with respect to aminoacyl-tRNA synt,hetases in this work allowed us t’o convert the enzyme with two act,ive centres into a one-site enzyme. This conversion creat,es Ned possibilities for further investigation of the enzyme by means of kinetic and other methods. All the data obtained here are summarized in the scheme presented in Figure 15. It may be seen that the functionally one-site enzyme map be obtained by one of

AFFINITY

LARELLING

OF

Trp-‘RN.1

STKTHETASE

498

Flu. 15. A scheme summarizing the affinity modification of TRSasc, by means of azido-ATl’ Cl-R-Trp-tRN.4. Each circle signifies 1 of the 2 subunits of the enzyme. ATP-binding sites are shown as hatched circles. triangles represent ATP, lines represent, chemical bonds. The non-specific binding of th(x azido-ATP outside the active sites is not shown. Stage 1. Initial (non-modified) TRSase. Stage 2. Photochemical addition of azido-ATP leading to complete blocking of ATP-binding &es and, hence, to complete inart,ivation both of ATI’ [32P]pyrophosphate exchange and of tR?r‘A aminoacylat,ion reactions. of R-Trp-tRNA is covalently Stage 3. TRSase subjected to complete alkylation, i.e. 1 mol bound to 1 mol of dimeric enzyme molecule. The aminoacylation activity is almost) completel? blocked since the binding of one of the tRNA molecules prohibit,s the binding of the second ow to the free tRN.4.binding site. The enzyme is partially active in t,he amino acid activat,ion reaction probably due to preservation of activity in the non-modified subunit. Stage 4. Alkylated enzyme modified with azido-ATP. No activity in either reaction since OIIV of the active sites is blocked by azido-ATP, and the other one by R-Trp-tRNA. Stage 5. Alkaline hydrolysis of the enzyme (stage 4) to split tRNA. The enzymatic activity in both reactions is partially restored dutt to liberation of onp of the active crntres previously blockrtl by R-Trp-t,RNA. Stage 6. Covalent binding of azido-ATE’ to the ATP-bintling crntrrs liberated aftcxr tho removal of t,he tRNA moictt,y. The enzyme in inactive in b&h reactions. and

two ways, either after the reaction with Cl-R-Trp-tRKA (form 3), or by sequential alkylation and photoaddition followed by removal of the tRNA moiety after alkalintb hydrolysis (form 5). In the first case the enzyme is not only in the one-site form but is also monofunctional: it catalyses the amino acid activation but does not catalyse tryptophanyltRNA synthesis within a reasonable tRNA concentration range. In the second case the enzyme remains bifunctional, i.e. it catalyses both ATP-[32P]pyrophosphat,e exchange and aminoacylation reactions but has only one active centre, as in the first case. Presumably, it may be of interest to compare the kinet’ic properties of thescb t w-0 forms. It is possible that the procedure developed might be applied to the other enzymes of t,he same group as well, and where there exist appropriate a,ffinity reagents it might also be used with other multimeric enzymes possessing the same properties. The observation that the covalent attachment of R-Trp-tRNA at one of the two active centres prevents the photochemical reaction of this (but not the other) centrc with azido-ATP may presumably be explained as taking place via spatial shielding of the ATP binding site with a covalent “bridge” between the enzyme and tRNA molecules, because after the removal of tRNA the reaction with azido-ATP proceeds without any difficulty. In essence it means that tRNA which is in complex wit#h

500

V.

Z.

;\KHVERDYAN

ET

.-1L.

TRSase prevents the formation of E-azido-ATP complex at one of thcl crntrcs. :IS if causing orientatjion in the direction of t,hr non-occupied cerurc. :\s a result, thts formation of adenylate in the presencca of tRNA may occur at onI>, one of the csentres. However, tryptophan cannot be transferred to tRNA from adenylate hecause tRNA is absent in this act,ive centre due bo the negative co-operativity effect. The protection of TRSase against alkylat’ion in the presence of ATP and t,ryptophan needs to be discussed separately. Those groups of the protein molecule that react with Cl-R-Trp-tRNA are located outside the active centre. and consequently the protective effect is not connected with the possible involvement of these groups in the binding of the ligands which protect them from a.lkylation. Not only does ATP prevent alkylation but alkylation, too. prevents the photochemical reaction with azido-ATP. In other words, competitive relations exist between azido-ATP, ATP and Cl-R-Trp-tRNA although ATP does not prevent the binding of Cl-R-Trp-tRNA and evidently their sites of binding are different. Covalent fixation of R-Trp-tRNA may sterically hinder the accessibility of the ATP binding site, and thus prevent the photochemical reaction with azido-ATP. On the other hand, when the ATP binding site is occupied with a ligand the alkylation reaction may not proceed, due to the fact that the active ethylene immonium cation of Cl-R-Trp-tRNA is spatially removed from the corresponding functional group of TRSase. The alternative explanation may be to assume that the influence of the ligands is concerned not only with steric hindrance hut with conformational changes induced by the ligands. There are some published observations (Kukhanova et al., 1968 ; Yarus & Berg, 1969; Gozshkova & Lavrik, 1975; Gorshkova et ccl.. 1976) in favour of the latter proposition. We are grateful to Mrs G. Moussinova and Mrs T. Bozenko for their help tion of the manuscript. This work was presented to the European Molecular Biology Organizat,ion tRNA structure and function (August 1976, Sonderberg, Denmark).

in the preparaworkshop

on

REFERENCES Ankilova, V. N., Knorre, D. G., Kravchenko, V. V., Lavrik, 0. 1. & Nevinsky, G. A. (1975). PEBS Letters, 60, 172-175. Babkina, G. T., Zaritova, V. F. & Knorre, D. G. (1975). Bioorgun. Chem. I, 611-617 (Russian, Eng. summary). Backer, B. R. (1967). Design of Active Site-Directerl Irreversible Enzyme Inhibitors, John Wiley and Son, New York and London. Bartmann, P., Hanke, T., Hammer-Raber, B. & Holler, E. (1974a). Biochem. Biophys. Res. Commun. 60, 743-747. Bartmann, P., Hanke, T., Hammer-Raber, B. & Holler, E. (1974b). Biochemistry, 13, 4171-4175. Bartmann, P., Hanke, T. & Holler, E. (1975). Biochemistry, 14, 4777 4786. Bazhulina, N. P., Kirpichnikov, M. P., Morosov, Yu. V. & Sarin, F. A. (1974). MoZ. Photochem. 6, 43%55. Blanquet, S., Dessen, P. & Iwatsubo, M. (1976). J. Mol. Biol. 103, 765-784. Bochkareva, E. S., Budker, V. G., Girshovich, A. S., Knorre, D. G. & Teplova, N. M. (1973). &ioE. BioE. 7, 278-288 (Russian, Eng. summary). Bruton, C. J. & Hartley, B. S. (1970). J. Mol. Biol. 52, 165-178. Budker, V. G., Knorre, D. G., Kravchenko, V. V., Lavrik, 0. I., Novinsky, G. A. $ Teplova, N. M. (1974). FEBS Letters, 49, 1599162. Dorizzi, M., Labouesse, B. & Labouesse, J. (1971). Eur. J. Biochem. 19, 563-572. Ehresmann, B., Imbault, P. & Weil, J. H. (1973). Anal. Biochem. 54. 454-463.

AFFINITY

LABELLING

OF

Trp-tIlXA

SYPiTHETASE

50 1

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