BIOCHIMIE, 1973, 55, 1317-1318.

Hydrolysis of leucyl-tRNA ,by leucyl-tRNA synthetase. Emma MANTACHIAN,(*), Ernes t MALYGUINE (**) and Alan H. MEHLER (***).

.~:

Laboraloire de Biochimie du Ddoeloppement, lns t i tu t de Biologic Mol~culaire, 2, Place Jussieu, 75005 Paris.

(10-8-1973).

INTRODUCTION.

Hydrolys i s of isoleucyl- tRNA by the corresponding syn the t a se f rom E. colt was sys temat ica l ly s tudied by Schreier and Schimmel I l l , who es tab l i shed tha t th is react ion proceeds s lowly ( turnover n u m b e r 0.8 per m i n u t e at pH 7 and 37 ° ) and tha t the dependence upon Mg ÷+ concent ra t ion and the influences of other subs t ra tes d i s t ingu i shed hydro lys i s f rom the esterifi- ca t ion of tRNA. Eldred and Schimmel [2] found t h a t the hydro lys i s reac t ion proceeds m u c h fas ter with valyl- tRNAne but t h a t phenylalanyl- tRNAHe is not split. Bonnet , Gieg6 and Ebel (3] found both valyl- aud pheny la l any l - tRNA syn the ta ses of yeas t to at tack m a n y of the homologous anainoacyl-tRNA species and also mischarged tRNAVal and tRNAPhe.

The existence of a hydro ly t ic reac t ion ra ises bo th biological ques t ions about the significance of th is appa ren t ly cont radic tory or se l f -defea t ing react ion, d iscussed by Schimmel and h is associates, and ques t ions of enzyme m e c h a n i s m [1, 3]. The i so la t ion of two f o r m s of teucyl- tRNA syn the t a se f rom E. coli, both of w h i c h recognize and bind tRNALeu bu t only one of which catalyzes the f o r m a t i o n of leucyl-tRNA [4], provided an oppor tun i ty to gain addi t ional ins igh t into the r e l a t ionsh ip be tween the hydro ly t i c and este- rification react ions.

MATERIALS AND METHODS.

Native and modified leueyl- tRNA syn the t a se s were separa ted f rom extracts of E. colt by the ch romato - graphic procedures of Rouget and Chapevil le [4]. The s ta r t ing ma te r i a l -was the appropr ia te f rac t ion f rom a large scale p repara t ion f rom w h i c h m e t h i o n y l - and pheny la l any l - tRNA syn the tases had been removed by gel f i l t ra t ion on Sephadex G-200. This ma te r i a l was generous ly suppl ied by Dr. J. P. Waller . The prote in precipi ta ted wi th a m m o n i u m sul fa te (50 g : 100 ml ; 70 p. cent sa tura t ion) was collected by eent r i fugat ion , dissolved in 0.025 M p o t a s s i u m phospha te buffer, pH 6.8, and dialyzed aga ins t the same buffer before

Supported by g ran t n ° 73.7.1191 f rom the DSldgation G6n6rale h la Recherche Scientifique et Technique.

(*) Recipient o.f a feilo:wship f rom the French government . P resen t address Katedra bioehimii , Biolo- g i tchesky facultet , Gosudar s t r eny Univers i te t , Erevan, Armenie, U.R.S.S.

(**) Recipient of a fe l lowship f rom the French government . Present address In s t i t u t Organi tcheskoy Chimii Sibirskovo, Otdeleniya Academii Naouk SSR, Novosibi rsk - - 90, U.R.S.S.

(***) J o h n Simon Guggenbeim Memorial Fellow. Presen t address D e p a r t m e n t of Biochemis t ry , Medical College of Wiscons in , Milwaukee, Wiscons in 53233, U.S.A.

being placed on a DEAE cellulose c o l u mn and e]uted wi th a gradient of phospha te buffer. The two peaks of leuc ine-dependent ATP-PP exchange act ivi ty (I and II) 'were f u r t h e r purified on c o l u mn s of hydroxy l - apat i te and the active f rac t ions f rom each r u n were pooled, precipi ta ted wi th a m m o n i u m sul fa te and dis- solved in 0.025 M p o t a s s i u m phosphate , pH 6.8. Peak II had specific act ivi t ies of 320 and 340 u/nag in the exchange and ester if icat ion react ions, respectively. Peak I had a specific act ivi ty of 300 u / r a g in the exchange react ion and no abi l i ty to fo rm leueyl-tRNA. ~4C Leucyl-tRNA was syn thes ized according to Rouget and Chapevil le [4]. Hydro lys i s of leucyl- tRNA was me a s u r e d as the ra te of decrease of ac id- insoluble radioact ivi ty .

RESULTS.

The pa r t i a l ly purified nat ive leucine act ivat ing enzyme was found to hydrolyze leucyl-tRNA. As had been f o u n d for other aminoacyl - tRNA syn the t a se s

1500 _

Z

500

?

O. 5 10 15

T i m e (min) Compar i son of two fo rms of Leucyl- tRNA syn the ta se

in the hyd ro lys i s of ]eucvl-tRNA. In each sample in a total vo lume of 1.0 ml Were 0.05 M Tris-HC1, pH 7.8 ; 0.05 M MgCl_. ; 2.10-5 M unf rac t iona ted tRNA contai- ning about 10 pnaoIes [14C]leucyl-tRNA ; and enzyme. Curve 1 represents a control w i t h o u t enzyme ; curve 2, 150 ,~g of enzyme f r o m Peak I ; curves 3, 4 and 5 sho~" the reac t ions wi th 5, 10 and 20 v,g, respect ively, of enzyme f rom Peak II.

f r o m E. colt and yeast , the reac t ion is about two orders of m a g n i t u d e s lower t h a n anainoacyl-tRNA f o r ma t i o n b u t it appears to be a func t ion of the same enzyme. Subs t i tu t ion of valyl - tRNA gave no hyd ro lys i s wi th

1 3 1 8 E. M a n t a c h i a n , E . M a l g g u i n e and A. H. Mehler .

th is p repara t ion and a homogeneous p repara t ion of the enzyme (Malyguine, Chapevil le and Mehler, sub- mit red for publ icat ion) ~vith a five-fold greater specific ac t iv i ty sho~ved a propor t iona l increase in the ra te of hydro lys i s of leucyl-tRNA.

The condi t ions for m a x i m u m ra tes of hydro lys i s are s imi la r to those found for the hydro ly t ic reac t ions of o ther aminoacy l - tRNA syn the ta ses and different f r o m the o p t i m u m condi t ions for the aminoacy la t i on of tRNA. Little react ion was observed at pH 6.5 or below ; be tween pH 7.0 and 7.8 the ra te of hydro lys i s was propor t iona l to enzyme concent ra t ion and independen t of pH. M a x i m u m rates of hydro lys i s 'were obta ined between 0.01 and 0.05 M MgClo ; 0.1 M MgC12 inhib i ted the ra te and EDTA e l imina ted the enzymat ic react ion. Under o p t i m u m condi t ions for the hydro lys i s of leucyl- tRNA by Peak II, Peak I fai led to catalyze a n y hydro lys i s .

After l earn ing f rom Dr. J. Bonnet (personal c o m m u - nication) t h a t he had found t h a t the cor responding hydroxyacy l - tRNA (produced by HNO, t r e a t m e n t of amiuoacyl - tRNA) could be hydro lyzed (albeit very slowly) by the valyl - tRNA syn the t a se of yeast , 'we deamina t ed 14C leucyl- tRNA by the method of Herv6 and Chapevil le [5] and found tha t th is hydroxyacy l - tRNA also cou]d be hydro lyzed enzymat i ca l ly by leucyt- tRNA syn the tase , at about one t en th the rate of leucyl-tRNA.

DISCUSSION.

Al though Rouget and Chapevil]e [6] reported the abi l i ty of both fo rms of the leucine ac t ivat ing enzyme to b ind tRNALeu, only the enzyme capable of cata- lyzing the t r a n s f e r react ion is capable of hydro lyz ing leucyl-tRNA. It seems likely, therefore, t h a t the hydro ly t ic react ion uses at least par t of the s t ruc tu res t h a t catalyze the t r a n s f e r reaction. Since the t r an s f e r func t ion can be res tored by the addi t ion of a pept ide to the modified enzyme [6], an exper imen ta l m e a n s

m a y be found for iden t i fy ing the roles of ind iv idua l a m i n o acid res idues in ca ta lyz ing par t s of the complex react ion t h a t produces aminoaeyl - tRNA.

The ab i l i ty of the enzyme to hydro lyse hydro- xyacyl- tRNA, a l though th i s type of compound does not par t ic ipa te in the reverse react ion wi th AMP and pyro- phosphate , suggests t h a t the hydro ly t ic m e c h a n i s m involves only a l imi ted in te rac t ion of the enzyme wi th the acylated tRNA and t h a t ac t iva t ion of the ester bond is a func t ion of the p rox imi ty of a group in the enzyme ; the act ivated ester t h e n reacts spon- t aneous ly wi th OH-. The re la t ive ly greater react iv i ty of esters of amino acids compared w i t h those of hydroxyac ids is reflected in the rates of enzymat i c hydrolys is . The repor t by Yarus [7] t h a t the pheny l - a l any l tRNA syn the tase of E. coli hydro lyzes isoleucyl- tRNAPhe rap id ly bu t not the corresponding deamina t ed produc t is no t necessar i ly in conflict w i t h th i s sugges- t ion as a general m e c h a n i s m . It is possible t h a t the very rap id hyd ro lys i s observed by Yarus involves a more e laborate catalyt ic m e c h a n i s m specific for an a mi n o acid and t h a t the smal l a m o u n t of enzyme used in his exper imen t s did not permi t d e m o n s t r a t i o n of the slow hydro lys i s of the hydroxyac id .

BIBLIOGRAPHY.

1. Schreir, A. A. & Schimmel , P. R. (1972) Biochemistry, l l , 1582-1589.

2. Eldred, E. W. & Schimmel , P. R. (1972) J. Biol. Chem., 247, 2961-2964.

3. Bonnet, J., Gieg6, R. & Ebel, J. P. (1972) FEBS Letters, 27, 139-144.

4. Rouget, P. & Chapeville, F. (1970) Ezzr. J. Biochem., 14, 498-508.

5. Herv6, G. & Cbapeville, F. (1965) J. Mol. Biol., 13, 757-766.

6. Rouget, P. & Chapevil le, F. (1971) Eur. J. Biochem., 23, 443-451.

7. Yarus, M. (1972) Proc. Nat. Acad. Sci. U. S., 69, 1915-1919.

BIOCH1MIE, 1973, 55, n ° 10.