11
286 BIOCHIMICA ET BIOPHYSICA ACTA BBA 96246 PEPTIDYL-tRNA VII. SUBSTRATE SPECIFICITY OF PEPTIDYL-tRNA HYDROLASE N. DE GROOT, Y. GRONER AND Y. LAPIDOT Department o[ Biological Chemistry, The Hebrew University o/ Jerusalen, Jerusalem (Israel) (Received March 4th, 1969) SUMMARY The enzymatic hydrolysis of peptidyl-tRNA is described. The hydrolysis rates of peptidyl-tRNA with different peptide chain lengths containing free and blocked 0t-amino groups are compared to the hydrolysis rates of N-acylaminoacyl-tRNA. It is shown that the hydrolysis rate of peptidyl-tRNA containing two peptide bonds is considerably higher than that of N-acylaminoacyl-tRNA. Moreover, the hydrolysis rate of different peptidyl-tRNA's depends on the peptide chain length. Thus, Gly2- Phe-tRNA is hydrolyzed faster than GlyPhe-tRNA, and Gly4Phe-tRNA is hydro- lyzed faster than Gly~Phe-tRNA. The apparent Km and Vmax values for Ac-Leu-tRNA are compared to those of Ala2Leu-tRNA. INTRODUCTION An enzyme which hydrolyzes N-acetylaminoacyl-tRNA was described by sev- eral groups 1-4. This enzyme, isolated from an Escherichia coli supernatant, hydro- lyzes the ester linkage between the N-blocked amino acid and the tRNA and does not hydrolyze unblocked aminoacyl-tRNA. It was found that the hydrolysis rate depends on the nature of the blocked amino acid attached to the tRNA. N-Ac-Val- tRNA, for example, was hydrolyzed at a slower rate than N-Ac-Ala-tRNA. In the present communication, the enzymatic hydrolysis of oligopeptidyl-tRNA is reported. The hydrolysis rates of peptidyl-tRNA with different peptide chain lengths containing free and blocked ~-amino groups are compared to the hydrolysis rate of N-acylaminoacyl-tRNA. The hydrolysis rate of peptidyl-tRNA containing at least two peptide bonds is higher than that of N-acetylaminoacyl-tRNA. Moreover, it is shown that the hydrolysis rate of different N-acylaminoacyl-tRNA's depends only to a small extent on the chain length of the acyl group. A preliminary report of this work has been published ~. MATERIALS AND METHODS [l*C]Amino acids were purchased from the Radiochemical Centre, Amersham, England and had the following specific activities (mC/mmole): 495 phenylalanine; 162 alanine; 311 leucine; 27° valine, tRNA E. coli B was obtained from Calbio- chem. Peptides were either purchased (from Miles-Yeda, Rehovot, Israel or from Cyclo, Chemical Co., Calif. U.S.) or prepared by condensing the N-hydroxysuccini- Bioehim. Biophys. Acta, 186 (1969) 286-296

Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

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Page 1: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

286 BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96246

PEPTIDYL-tRNA

VII. SUBSTRATE SPECIFICITY OF PEPTIDYL-tRNA HYDROLASE

N. D E G R O O T , Y. G R O N E R AND Y. L A P I D O T

Department o[ Biological Chemistry, The Hebrew University o/ Jerusalen, Jerusalem (Israel) (Received March 4th, 1969)

SUMMARY

The enzymatic hydrolysis of peptidyl-tRNA is described. The hydrolysis rates of peptidyl-tRNA with different peptide chain lengths containing free and blocked 0t-amino groups are compared to the hydrolysis rates of N-acylaminoacyl-tRNA. It is shown that the hydrolysis rate of peptidyl-tRNA containing two peptide bonds is considerably higher than that of N-acylaminoacyl-tRNA. Moreover, the hydrolysis rate of different peptidyl-tRNA's depends on the peptide chain length. Thus, Gly 2- Phe-tRNA is hydrolyzed faster than GlyPhe-tRNA, and Gly4Phe-tRNA is hydro- lyzed faster than Gly~Phe-tRNA.

The apparent Km and Vmax values for Ac-Leu-tRNA are compared to those of Ala2Leu-tRNA.

INTRODUCTION

An enzyme which hydrolyzes N-acetylaminoacyl-tRNA was described by sev- eral groups 1-4. This enzyme, isolated from an Escherichia coli supernatant, hydro- lyzes the ester linkage between the N-blocked amino acid and the tRNA and does not hydrolyze unblocked aminoacyl-tRNA. It was found that the hydrolysis rate depends on the nature of the blocked amino acid attached to the tRNA. N-Ac-Val- tRNA, for example, was hydrolyzed at a slower rate than N-Ac-Ala-tRNA.

In the present communication, the enzymatic hydrolysis of oligopeptidyl-tRNA is reported. The hydrolysis rates of peptidyl-tRNA with different peptide chain lengths containing free and blocked ~-amino groups are compared to the hydrolysis rate of N-acylaminoacyl-tRNA. The hydrolysis rate of peptidyl-tRNA containing at least two peptide bonds is higher than that of N-acetylaminoacyl-tRNA. Moreover, it is shown that the hydrolysis rate of different N-acylaminoacyl-tRNA's depends only to a small extent on the chain length of the acyl group.

A preliminary report of this work has been published ~.

MATERIALS AND METHODS

[l*C]Amino acids were purchased from the Radiochemical Centre, Amersham, England and had the following specific activities (mC/mmole): 495 phenylalanine; 162 alanine; 311 leucine; 27 ° valine, tRNA E. coli B was obtained from Calbio- chem. Peptides were either purchased (from Miles-Yeda, Rehovot, Israel or from Cyclo, Chemical Co., Calif. U.S.) or prepared by condensing the N-hydroxysuccini-

Bioehim. Biophys. Acta, 186 (1969) 286-296

Page 2: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

PEPTIDYL-tRNA HYDROLASE 287

mide ester of N-carbobenzoxyamino acid with p-nitrobenzyl ester of another amino acid or peptide according to methods described in the literature 6,~. The protecting groups were removed after condensation by catalytic hydrogenation.

[14ClAminoacyl-tRNA's were prepared according to NATHANS AND LIPMANN s. For the preparation of [zaC~Val-tRNA, we used the purified Val-tRNA synthetase prepared according to BERGMANN et al. °.

Peptidyl-tRNA's were prepared by condensing N-hydroxysuccinimide esters of N-monomethoxytri tylamino acid or peptide with [14Claminoacyl-tRNA and by subsequently removing the monomethoxytri tyl group by treating the N-blocked pep- tidyl-tRNA with dichloroacetic acid ~°-12. In this work, all the acylation reactions were carried out in a mixture of freshly distilled 80 % dimethylsulfoxide and o.I M 20 % acetate buffer (pH 5.0). Gly4Phe-tRNA and Gly~Phe-tRNA were prepared by the stepwise technique (details will be published elsewhere).

Acylaminoacyl-tRNA's were prepared by acylation of the amino-acyl-tRNA using N-hydroxysuccinimide ester of the appropriate fa t ty acid as the acylating agent by a modification of the method reported previously ~3. 2 mg [14C3Phe-tRNA (250 ooo counts/rain per rag) dissolved in 0.5 ml of o.1 M acetate buffer (pH 5.0) were added to a solution of 30 mg N-hydroxysuccinimide ester of acetic acid (187 /Jmoles) in 2.0 ml of freshly distilled dimethylsulfoxide. The reaction mixture was kept at 3 °0 for 16 h, and the tRNA was precipitated by adding I vol. of IO °/o di- chloroacetic acid. The precipitate was centrifuged and washed three times with cold absolute ethanol. The dry precipitate was dissolved in o.i M acetate buffer (pH 5.0). An aliquot was treated with 0.5 M NaOH for I h at 3 o°, and the hydrolysate was analyzed by paper chromatography. All radioactivity moved as Ac-Phe and none as the free amino acid.

The same procedure was used for the preparation of Ac-I~C~aminoacyl-tRNA other than Phe-tRNA, formylI14C~aminoacyl-tRNA, caproyl-[14C~Phe-tRNA and lauroyl- II*C~Phe-tRNA.

Acetylpeptidyl-tRNA's such as Ac-Gly[14C~Phe-tRNA and Ac-GlyGly~laC~ - Phe-tRNA were prepared by acetylation of the unblocked peptidyl-tRNA using N- hydroxysuccinimide ester of acetic acid as described above.

Paper electrophoresis and paper chromatography Paper electropboresis was performed in a 45oo-V apparatus in which What-

man 3 MM paper was immersed in a water-cooled high boiling petroleum fraction (Versol). The solvents used were: I M acetic acid (pH 2.5) and acetic acid-formic acid-water (3 : I : 16, by vol., pH 1.9).

Paper chromatography was performed by the descending technique using Whatman No. I paper. The solvent used was n-butanol-acetic acid-water (78 : 5 : 17, by vol.).

Amino acids and peptides were detected by ninhydrin; N-blocked amino acids or peptides were detected by spraying the chromatogram with a solution containing IO mg bromocresol green and 0. 5 ml formaldehyde in IOO ml acetone. The N-blocked amino acids became yellow 14. When radioactive compounds were used the electro° phoretogram or chromatogram was cut into 1.5-cm wide strips, and the radioactivity was determined in a Packard liquid scintillation counter by immersing the pieces of paper in a glass vial containing IO ml of scintillation liquid.

Biochim. Biophys. Acta, 186 (1969) 286-296

Page 3: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

288 N. DE GROOT et al.

Preparation o/peptidyl-tRNA hydrolase A. Unpuri]ied enzyme. Dialyzed 15o ooo × g centrifuged supernatant (I5o-S frac-

tion) was prepared from E. coli W essentially as described by NIRENBERG 15. The supernatant was analyzed for peptidyl- tRNA hydrolase activity and is referred to as the "unpurified enzyme".

B. Purified enzyme. A 32-ml portion of the I5o-S fraction (576 mg protein) was applied to a DEAE-cellulose column (2.8 cm × lO.5 cm) previously equilibrated with Buffer A containing o.oi M Tris acetate (pH 7.5), o.oi M magnesium acetate, o.oi M KC1, o.oi M mercaptoethanol and lO -4 M EDTA. The column was eluted stepwise with Buffer A containing various concentrations of KC1 (Fig. I). The eluate was col- lected in Io-ml fractions which were analyzed for enzyme activity. The protein con- tent was determined according to LOWRY et al. 16. Fractions 3-12 contained the en- zymatic activity of pept idyl- tRNA hydrolase. This fraction is referred to as "puri- fied enzyme".

All extraction and purification operations were carried out at 4 ° . Both the unpurified and purified enzymes could be stored at --20 ° for at least

5 months without loss of activity.

Assay ]or enzymatic activity The assay mixtures contained o.I M Tris acetate (pH 8.0) and o.ooi M mag-

nesium acetate. The various amounts of enzyme and radioactive substrates used are given in the legends to the figures. To assays not containing enzyme, the same vol- umes of Buffer A were added. All incubations were carried out at 3 o°. The reactions were star ted by adding the substrates; aliquots were removed at various time inter- vals and assayed for residual trichloroacetic aid-insoluble radioactivity ~.

In the case of lauroyl-E14C~ Phe-tRNA, the trichloroacetic acid precipitation tech- nicque could not be used because the free lauroyl-Phe precipitated together with the lauroyl-Phe-tRNA. Therefore, aliquots of the reaction mixture were taken at various time intervals and were added to test tubes which contained 0. 5 ml of o.I M potas- sium acetate buffer (pH 5.0) and 1.5 ml of ethylacetate. Each test tube was shaken in a vortex mixer for 30 sec and the two layers were separated by centrifugation. A I.o-ml aliquot was taken from the organic layer, and its radioactivity was determined after adding it to IO ml of scintillation liquid prepared according to BRAY 17.

Analyzes of the degradation products were performed by high voltage paper electrophoresis and paper chromatography together with the appropriate markers.

All the aminoacyl- tRNA derivatives are hydrolyzed spontaneously at the pH of the enzymatic reaction (o.I M Tris-acetate, pH 8.0). This reaction is a first-order one. The hydrolysis observed in the presence of the peptidyl- tRNA hydrolase, which includes the nonenzymatic hydrolysis, is also a first-order reaction. (Plots of In c versus time give straight lines). From the hydrolysis rates in the absence and presence of enzyme, we can calculate the first-order kinetic constants of both the nonenzymat- ic and enzymatic hydrolyzes. The first-order kinetic constant for the enzymatic hydrolysis is equal to the difference between the two constants:

/~enz ~ kobs--knonenz

in which kenz ~--- calculated kinetic constant for the enzymatic hydrolysis, kobs ----- ki- netic constant for the hydrolysis obtained in the presence of enzyme and knonenz kinetic constant for the nonenzymatic hydrolysis.

Biochim. Biophys. ,dcta, 186 (1969) 286-296

Page 4: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

P E P T I D Y L - t R N A HYDROLASE 289

Using kenz, the percentage of hydrolysis at the different time intervals due only to the enzymatic reaction was calculated according to: Percentage of substrate hydrolyzed = (I--e-~t)IOO. All the hydrolysis rates presented in this paper were cor- rected according to this procedure.

RESULTS AND DISCUSSION

As can be seen from Fig. I, nearly all the peptidyl-tRNA hydrolase is eluted from the DEAE-cellulose column by o.oi M KC1. The increase in the specific activity is about I5-2o-fold in this step. The partially purified hydrolase is free of enzymatic activities which can interfere with the assay of the hydrolase reaction.

E v

Z

! /\ "!\

50 100 150 200 250 3O0 ML

O.01M 'fl,~. 3 . , M - ' ~ O . 2 M ~ ~).SM--'~ K C L

30

20

z

,o i

Fig. I. Pur i f i ca t ion of p e p t i d y l - t R N A hydro l a se b y DEAE-ce l lu lose c h r o m a t o g r a p h y . . 3 2 ml (576 m g pro te in) of 15o ooo × g cen t r i fuged s u p e r n a t a n t f rac t ion ob t a ined f rom E. coli was appl ied to a c o l u m n (2.8 cm × 1o.5 cm) equ i l ib ra ted w i t h Buf fe r A (o.oi M "Iris ace t a t e (pH 7.5), o .o i M m a g n e s i u m ace ta te , o .o i M KC1, o .o i M m e r c a p t o e t h a n o l and io-* M E D T A ) . The c o l u m n was e lu ted s tepwise w i th Buf fe r A c o n t a i n i n g inc reas ing concen t r a t i ons of KCI. i o -ml f rac t ions were collected. One u n i t of pept idyl- tRl ' f fA hydro lase is de f ined as the a m o u n t of e n z y m e caus ing t he hyd ro lys i s of IOOO c o u n t s / m i n Gly , [14C]Phe- tRNA per m i n a t 30 °.

(I) The hydrolase is free of ribonuclease GlyGly[14CIVal-tRNA was treated with the purified peptidyl hydrolase. After 65 % of the peptidyl-tRNA was hydrolyzed purified valyl-tRNA synthetase was added with [laClvaline. All the tRNA val liber- ated as the result of the hydrolysis of the GlyGlyE14CJVal-tRNA was recharged with [l~C]valine (Fig. 2).

As previously reported as, in tRNA val (yeast) at least one nucleotide bond can be cleaved without changing the amino acid acceptance capacity; thus the above mentioned experiment was repeated using GlyGly[14C]Leu-tRNA as substrate. After 85 % of the substrate was hydrolyzed, it could be IOO % recharged. It seems, there- fore, that our peptidyl-tRNA hydrolase is practically free of ribonuclease.

(2) The hydrolase is free of peptidase. The unpurified enzyme acted on GlyGly- [I*C]Phe-tRNA, and after incubation the reaction mixture was analyzed by high voltage paper electrophoresis. As shown in Fig. 3 A, the major radioactive product

Biochim. Biophys. Acta, I86 (1969) 286-296

Page 5: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

290 N. DE GROOT et al.

t~ (3

2ooo

,~ \ -.'~OROL,SE

Z

(~ ~ _ +HYDROL ASE

T,*~ (*,N) l ADDITION OF

CHt-RGING M~XTURE

Fig. 2. Charging capaci ty of tRNAVal after part ial hydrolysis of Gly~Val-tRNA by pep t idy l - tRNA hydrolase. The 0.5 ml incubat ion mix ture contained 36 ooo counts /min Gly~[14C]Val-tRNA and the enzyme (2o/~g protein) . 25/~1 aliquots were removed at o, 5, io, 15 and 20 rain and were assay- ed for acid-insoluble radioact ivi ty as described in MATERIALS ANn METHODS. At 20 min after incu- bation, the react ion mixture was chilled in an ice ba th and 5/~1 of o.25/~C ~14Clvaline, of o.I M ATP, of I M acetic acid and of purified E. coli valy l - tRNA synthe tase were added. The incubat ion mix ture was b rough t to 37 ° and 25-/,1 samples were removed at various t ime intervals for measurement of radioact ivi ty precipi tated in acid.

was [14C]phenylalanine and only a small amount of Gly-Gly-Phe could be detected. When the purified hydrolase was used, Gly-Gly-Phe was the sole radioactive product formed (Fig. 3B). Similar results were obtained using GlyGIy[14C]Val-tRNA and AlaAlaE14C]Ala-tRNA as substrates. Our findings, however, do not exclude the possi- bility of peptidase activity in the purified hydrolase preparation, which attacks pep- tide bonds other than those of glycine and alanine peptides.

(3) The peptidyl-tRNA hydrolase attacks Phe-tRNA and Val-tRNA very slow- ly, if at all. After incubation of E14C~Phe-tRNA or E14C~Val-tRNA with the unpurified enzyme (SI5o) and after precipitation of the tRNA by trichloroacetic acid, some radio- activity was found in the trichloroacetic acid-soluble fraction (Figs. 4A and 4B). This hydrolytic activity was enhanced by a rather high (0.03 M) Mg *+ concentration. The products liberated were proved by paper electrophoresis to be phenylalanine and va- line respectively. The purified peptidyl-tRNA hydrolase, however, does not attack the above-mentioned aminoacyl-tRNA's even in the presence of 0.03 M magnesium ace- tate (Figs. 4A and 4B) and acts only on N-substituted aminoacyl-tRNA's. It is in- teresting to note that the action of the unpurified enzyme on aminoacyl-tRNA is dependent on the source of the tRNA. Phe-tRNA from yeast was not attacked by the unpurified enzyme. The peptidyl-tRNA hydrolase, on the other hand, hydrolyzed GlyGlyI14ClPhe-tRNA prepared from yeast tRNA at the same rate as GlyGly[14C~- Phe-tRNA prepared from E. coli tRNA (Fig. 5).

During the purification of peptidyl-tRNA hydrolase by a DEAE-cellulose col- umn, the activity which hydrolyzes Phe-tRNA was clearly separated from the pep- tidyl-tRNA hydrolase.

Biochim. Biophys. Acta, 186 (1969) 286-296

Page 6: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

PEPTIDYL-tRNA HYDROLASE 291

-- 1 6 0 0

~ 160 o

> O

2 0 0 0 <

O

<

1 0 6

PHE G ~ H E

A

0 15 30

D I S T A N C E

4 5 6 0 75

F R O M O R I G I N ( C M

Fig. 3. Paper electrophoretic analysis of the enzymatic hydrolysis of Gly~[14C]Phe-tRNA. The 35 ~ul incubation mixture contained: 5ooo counts/min Glyz[14C]Phe-tRNA, o.o5 M Tris acetate (pH 8.o), o.ooi M magnesium acetate, unpurified enzyme (4 ° / , g protein (A) and purified enzyme ( i / ,g protein (B). 2o min after incubation at 3o% the total mixtures were subjected to paper electrophoresis in i M acetic acid on "Whatman No. 3 MM paper (pH 2.5) with the appropriate markers.

/r

i . / / /

5 10 20

T I M E I M * N )

o

o a- c~ >

o

o /

/

5 10 15 20 25 3O

Fig. 4. Kinetics of hydrolysis of E. coli [I4C]Phe-tRNA (A) and [14C]Val-tRNA (B) in the presence of purified peptidyl-tRNA hydrolase and in the presence of 15o ooo X g centrifuged supernatant at two different Mg 2+ concentrations. The o.25-ml reaction mixtures contained: o. I M Tris acetate (pH 8.0); [14C]Phe-tRNA (2o ooo counts/min), (A) or [14C]Val-tRNA (20000 counts/rain) (B); purified peptidyl- tRNA hydrolase (50/*g protein) or 15o ooo ×g supernatant (40 /~g protein). Aliquots of 25 /~i were removed at various time intervals for measurement of radioactivity precipitated in acid. For details see MATERIALS AND METHODS. [ ~ ] , 15o ooo×g supernatant hydrolase at 0.03 M 15o ooo ×g supernatant at o.oo3 M Mg~+; A - A , purified peptidyl-tRNA at 0.03 M Mg2+; I - I , Mg2+; & - A , purified peptidyl-tRNA hydrolase at o.oo 3 M Mg 2+.

Biochim. Biophys. Acta, 186 (1969) 286-296

Page 7: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

292 ~. DE GROOT et al.

1°o

2

sm

T I M E I M I N )

""-g.. . .

' ' ' I I ' , I I ' I I ' !C 25 50 1GO .~50 50.0 1000

Magnes ium ace ta te (m i ,A )

Fig. 5. Kinetics of hydrolysis of E. coli Gly,[z4C]Phe-tRNA and yeast GIy,[z4C]Phe-tRNA. The 25o-~ul reaction mixtures contained: o.I M Tris acetate (pH 8.o); o.ooi M magnesium acetate; Glyz[14C~Phe-tRNA (yeast or E. coli) (2o ooo counts/min); purified peptidyl-tRNA hydrolase (25/*g protein). 25-/~1 aliquots were removed at various time intervals for measurement of radio- activity precipitated in acid. ZX-/',, E. coli Gly 2 [14C~ Phe-tRNA; A - A, yeast Gly,[z4C]Phe-tRNA.

Fig. 6. Peptidyl- tRNA hydrolase activity as a function of Mg *+ concentration. Peptidyl-tRNA hydrolase (io/zg protein) was incubated for 2 rain with an amount of EDTA equimolar to that of the Mg 2+ content of the enzyme solution. The preincubated enzyme was added to a ISO-~Ul reaction mixture containing o.2 M Tris acetate (pH 8.o); Glyl[14C]Val-tRNA (io ooo counts/rain) and additional magnesium acetate as indicated. 3o-/,1 aliquots were removed at various time intervals for measurement of radioactivity precipitated in acid.

On preincubating the purified peptidyl-tRNA hydrolase at 4 ° for 2 rain with an amount of EDTA equivalent to that of the Mg 2+ present in the enzyme preparation all enzymatic activity was lost. Addition of magnesium acetate to an incubation mixture containing the EDTA-preincubated enzyme restored the hydrolytic activ-

s e •

-

/ . /

s l o 2o 3Q

T IME IM IN 1

Fig. 7. Kinetics of enzymatic hydrolysis of different N-acyl-E14C]Phe-tRNA's. The 225-#1 reaction mixtures contained: o.I M Tris acetate (pH 8,o) o.ooi M magnesium acetate; 2o ooo counts]rain of acyl-[x4C]Phe-tRNA (all the substrates were prepared from the same [z*C]Phe-tRNA prepara- tion); purified peptidyl-tRN A hydrolase (5 °/~g protein). 25-/zl aliquots were removed at various time intervals for measurement of radioactivity precipitated in acid. Lauroyl-[x*C]Phe-tRNA hydrolysis was determined by ethyl acetate extraction. Caproyl-Et'C]Phe-tRNA was determined by both the ethyl acetate extraction and the trichloroacetic acid precipitation methods. The two methods gave identical results. For details see MATERIALS AND METHODS. (D-(D, Gly,[X*C] Phe- tRNA; V]-[[], formyl-[x4C]Phe-tRNA; A - A , caproyl-[l*C]Phe-tRNA; A - & , lauroyl-[liC~Phe- tRNA; m-m, Ac-tX4C]Phe-tRNA.

Biochim. Biophys. Acta, 186 (1969) 286-296

Page 8: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

PEPTIDYL-tRN_A HYDROLASE 293

ity. Fig. 6 describes the dependence of hydrolase activity on the concentration of mag- nesium acetate which was added to the EDTA-preincubated enzyme. At lO -3 M Mg *+ a maximum is reached; at concentrations higher than IO -* M the enzymatic activity gradually decreases.

The hydrolysis rate of different acylaminoacyl-tRNA's was compared. As can be seen from Fig. 7, acyl-Phe-tRNA's which differ in the chain length of the acyl group, such as formyl and caproyl, were hydrolyzed at approximately the same rate. Formyl-Phe-tRNA, however, was hydrolyzed definitely faster than Ac-Phe-tRNA.

From Fig. 8A it can be concluded that substrates possessing at least two pep- tide bonds are hydrolyzed much faster than those substrates with only one peptide

l A

O

o

z 5O .--<

5 10 15 20 25 30

T I M f ( M IN )

Q uJ N >. ,J O ¢ O >- Z 50 / / / " J °

0 /

5 10 5 2O 25 O

T I M E (M IN )

Fig. 8. Relative enzymatic hydrolysis rates of peptidyl-tRNA's with different chain lengths. The 225-/,1 reaction mixtures contained: o.i M "iris acetate (pH 8.o); o.ooi M magnesium acetate; 20 ooo counts/min of Glyn[l*e]Phe-tRNA; purified peptidyl-tRNA hydrolase 25/zg protein (A) and 5/zg protein (13). 25-/,1 aliquots were removed at various time intervals for measurement of radioactivity precipitated in acid. For details see MATERIALS ANn METHODS. A. O - Q , Ac-Gly2- [liC]Phe-tRNA; A - A , Ac-[Gly[14C]Phe-tRNA; A - A , Gly2114C]Phe-tRNA; C)-O, Ac-[14C]Phe - tRNA; I t - I , Gly[14C]Phe-tRNA. B. / x -A , GIy6[I*C]Phe-tRNA; V - V , Gly4[~4C]Phe-tRNA; C7-U1, Gly,[14CJPhe-tRNA; 0 - 0 , Gly[IIC]Phe-tRNA; O-C), Ac-[I~C]Phe-tRNA.

Biochim. Biophys. Acta, 186 (1969) 286-296

Page 9: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

294 N. DE GROOT et al.

bond. GlyE14C~Phe-tRNA is hydrolyzed at a rate similar to that of Ac@4C~Phe-tRNA, but both GlyGly[14C]Phe-tRNA and Ac-GlyEI~CIPhe-tRNA are hydrolyzed consider- ably faster. A third peptide bond, as in Ac-GlyGly [14CIPhe-tRNA , seems to enhance the initial hydrolysis rate even more. Fig. 8B shows that Gly4E*4CIPhe-tRNA is also hydrolyzed faster than Gly2[t4CIPhe-tRNA. However, a substrate with a longer pep- tide chain, such as Glys~I~C]Phe-tRNA, is not hydrolyzed faster than Gly4I~4CIPhe- tRNA. It should be mentioned that the enzyme concentration used in the experiment described in Fig. 8A is much higher than the concentration used in the experiment described in Fig. 8B. Similar results were obtained with some p4C]Val-tRNA deriv- atives (Figs. 9 A and B). Formyl-II4CIVal-tRNA is hydrolyzed faster than Ac-E14CIVal - tRNA, but both are much poorer substrates than GlyGlyE14CIVal-tRNA.

100

0

I * i t I - - I 5 10 15 20 ~5 30

T I M E ( M t N )

B

>.

a

o: a

J /

/

S 10 1 5 2 0 2 5 3 0

T I M E ( M I N ]

Fig. 9. Kinet ics of e n z y m a t i c hydro lys i s of some [z¢C]VaI-tRNA derivat ives . The 225-#,1 reaction mixtures contained: o.I M Tris acetate (pH 8.o); o.ooi M magnes ium acetate; 20 ooo counts/rain of the different der ivat ives (all the substrates were prepared from the same [14C]Val-tRNA preparation); purified p e p t i d y l - t R N A hydro lase 50/~g protein (A) and 5/*g protein (B). 25-/,1 al iquots were removed at various t ime intervals for measurement of radioact iv i ty precipi tated in acid. For detai ls see MATERIALS AND METHODS. [3--O, Gly~[14CTVal-tRNA; I - U , formyl-~14C~ Val- tRNA; ~ - z ~ , A c - [ 1 4 C ] V a l - t R N A ; a t - A , [14C]Val-tRNA,

Biochim. Biophys. Acta, 186 (1969) 286-296

Page 10: Peptidyl-tRNA VII. Substrate specificity of peptidyl-tRNA hydrolase

P E P T I D Y L - t R N A HYDROLASE 295

c

N >-

0

1 @ O]

!

lOG

/ /

1'0 ~'5 2'0

T I M E

. / , /

. / /

,,/" /

I M I N I

Fig. io Kine t ics of e n z y m a t i c hydro lys i s of some [14C]AIa-tRNA and [14C]Leu-tRNA der iva t ives . The 225-1~1 reac t ion m i x t u r e s con ta ined : o . i M Tris ace ta te (pH 8.0); o .oo i M m a g n e s i u m ace ta te ; 20 ooo coun t s /mJn of t he d i f fe rent subs t r a t e s ; pur i f ied p e p t i d y l - t R N A hydro lase (5/zg prot(,in). 25-/~1 a l iquots were r e m o v e d a t va r ious t ime in t e rva l s for m e a s u r e m e n t of r ad ioac t iv i ty precipi- t a t e d in acid. For deta i l s see MATERIALS AND METHODS. V]-V],Ala,[I*C]Ala-tRNA; A - A , Ac-[14Cj- A l a - t R N A ; A - A , [14C]Ala-tRNA; I - @ , Gly2?4C]Leu- tRNA; Q-(Z), Ac-[14C]Leu-tRNA; O - O , [14C]Leu-tRNA.

Figs. IoA and B show that in the case of leucyl-tRNA and alanyl-tRNA deriv- atives, the peptidyl-tRNA with two peptide bonds is hydrolyzed significantly faster than the corresponding acetyl derivatives. Some of our results somewhat contradict those obtained by PAULIN et al . 10 who reported that Ac-Val-tRNA is hydrolyzed faster than formyl-Val-tRNA. Moreover, they found similar hydrolysis rates for Ac-

,/v

i/v s / 4 4

2 2

, * " d

,/fs},,~M-,~ ,/~]2,,,(,,-,~ Fig. I I. L i n e w e a v e r - B u r k plot (I/[S]versus I/V) for Ac-[14C]Leu-tRNA. The IOO-/zl r eac t ion mix- tu re contaAned: o . i M Tris ace t a t e (pH 8.o), o .oo i M m a g n e s i u m ace t a t e and pur i f ied hydro lase (5 l*g prote in) . The s u b s t r a t e concen t r a t i on as indica ted , A t eve ry s u b s t r a t e concen t r a t ion a l iquots were r e m o v e d a t d i f fe rent t i me in t e rva l s (I, 2, 3 and 5 min) and t he ac id-precipi table radio- a c t i v i t y de te rmined . F r o m these resu l t s the ini t ia l ve loc i ty r a t e s were t a k e n for t he L i n e w e a v e r - B u r k plot. Veloci ty expressed in pmo l e s s u b s t r a t e hyd ro lyzed per io rain.

Fig. 12. L i n e w e a v e r - B u r k plot (z/IS]versus I/v) for Ala , [ I*C]Leu- tRNA. For reac t ion cond i t ions see Fig. I I . Veloc i ty expressed in pmo l e s s u b s t r a t e hyd ro lyzed per IO rain.

Biochim. Biophys. Acta, 186 (I969) 286-296

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296 N. DE GROOT et al.

Phe- tRNA and the N-blocked pept idyl- tRNA Ac-PhePhe-tRNA. According to our results the hydrolysis rate of Ac-GlyPhe-tRNA was much faster than that of Ac- Phe-tRNA. Therefore, it seems that the number of peptide bonds rather than the position of the terminal amino group being free or blocked determines the enzymatic hydrolysis rate.

We have calculated and compared the apparent K,~ and Vm~x values for an acyl- aminoacyl- tRNA such as Ac-E14ClLeu-tRNA and for a corresponding peptidyl- tRNA such as AlaAlaE14CILeu-tRNA possessing two peptide bonds. Our preparations of Ex4cl- aminoacyl-tRNA derivatives contain, besides the labeled substrates, uncharged tRNA. CuzI~ et al. 1 showed that uncharged tRNA inhibits the hydrolase action. However as both Ex4C~leucyl-tRNA derivatives were prepared from the same [14C~leucyl-tRNA preparation, the results obtained have a comparative value. K m and Vmax calculated from the data of Fig. I I for Ac-[14CILeu-tRNA are 17 • lO -7 M and 0.5 pmole/min, respectively, and the K m and Vm~x for AlaAlaEI*CILeu-tRNA are 6. 7 • Io -7 M and 5.0 pmole/min, respectively (Fig. I2).

Our conclusion with respect to the substrate specificity is that the number of peptide bonds in the substrate is important . GlyPhe- tRNA which contains one peptide bond is hydrolyzed considerably slower than Gly2Phe-tRNA which contains two peptide bonds. Gly4Phe-tRNA which contains four peptide bonds is hydrolyzed definitely faster than Gly2Phe-tRNA. I t may be possible, however, that a maximal hydrolysis rate is reached with substrates possessing four peptide bonds, since Gly 8- Phe- tRNA is not hydrolyzed faster than Gly4Phe-tRNA. This conclusion however needs further experimental evidence. In any case it is clear from the results described in this paper that mere elongation of the substituent on the amino group of the aminoacyl- tRNA is not sufficient in itself to increase the hydrolysis rate of the pep- t idyl- tRNA hydrolase; formyl-Phe-tRNA, caproyl-Phe-tRNA and lauroyl-P he-tRNA were all hydrolyzed at approximately the same rate.

REFERENCES

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2 N. DE GROOT, A. PANET AND Y. LAPIDOT, Biochem. Biophys. Res. Commun., 31 (1968) 39. 3 H. KOSSEL AND U. I. RAJBHANDARY, J. Mol. Biol., 35 (1968) 539. 4 Z. VOGEL, A. ZAMIR AND D. ELSON, Proc. Natl. Acad. Sci., U.S., 61 (1968) 7Ol. 5 J- GRONER, N. DE GROOT AND Y. LAPIDOT, Israel J. Chem., 6 (1968) 96 p. 6 H. SCHWARZ AND K. ARAKAWA, J. Am. Chem. Soc., 81 (1959) 5691. 7 G. \¥. ANDERSON, J. ]~. ZIMMERMAN AND F. M. CALLAHAN, J. Am. Chem. Soc., 86 (1964) 1839. 8 D. lXTATHANS AND F. LIPMANN, Prov. Natl. Avad. Svi., U.S., 47 (1961) 467 . 9 1~'- H. BERGMANN, P. BERG AND M. DIECKMAN, J. Biol. Chem., 236 (1961) 1735.

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Biochim. Biophys. Acta, 186 (1969) 286-296