Inactivation of aminoacyl-tRNA Synthetases by amino acid chloromethylketones

77

Biochimica et Biophysica Acta, 340 (1974) 77--89 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 97929 INACTIVATION O F AMINOACYL-tRNA SYNTHETASES BY AMINO ACID CHLOROMETHYLKETONES*

JACK SILVER and RICHARD A. LAURSEN Department of Chemistry, Boston University, Boston, Mass. 02215 (U.S.A.)

(Received September 12th, 1973)

Summary Several amino acid chloromethylketones having the general structure R 0

+1 11

H3N-CH-C-CH:Cl were synthesized with the intent of labeling the active sites of the aminoacylt R N A synthetases. Of the c o m p o u n d s prepared, only the valyl chloromethylketone was sufficiently stable in pH 7.3 buffer for extensive inactivation studies. The L-valyl chloromethylketone caused little inactivation of the lysyl- and phenylalanyl-tRNA synthetases, but rapidly inactivated the valyl- and leucylt R N A synthetases. The amino acid acceptor activity of leucyl-tRNA synthetase was inhibited approximately ten times as rapidly as that of the valyl-tRNA synthetase. The inhibition appears to be irreversible since neither dialysis against buffer nor mercaptoethanol, which was found to destroy the inhibitor, caused reactivation of the enzymes. Both D-valyl chloromethylketone and chloroacetone were found to inhibit the aminoacyl-tRNA synthetases, although the rate of inactivation by chloroacetone was significantly lower than for the valyl analogs. Dissociation constants of complexes between valyl-tRNA synthetase and L- and D-valyl chloromethylketone were 20 and 100 mM, respectively. Chloroacetone did not exhibit saturation kinetics with this enzyme. With the leucylt R N A synthetases, L- and D-valyl chloromethylketone and chloroacetone were found to have dissociation constants of 7.5, 4.5, and 103 mM, respectively. The valyl- and leucyl-tRNA synthetases were protected from inactivation b y their natural substrates (cognate amino acid, ATP + Mg 2÷, or both). These data

* A preliminary r e p o r t o f part o f this w o r k w a s p r e s e n t e d at the 1 6 0 t h M e e t i n g o f t h e A m e r i c a n C h e m i c a l S o c i e t y , Chicago~ 1 9 7 0 ; B i o l o g i c a l A b s t r a c t N o . 2 0 0 .

78 suggest that L-valyl chloromethylketone inhibits b y reaction at the active sites of the valyl- and leucyl-tRNA synthetases.

Introduction

The aminoacyl-tRNA synthetases (L-amino-acid:tRNA ligase (AMP), EC 6.1.1.) are a group of enzymes catalyzing a two-step reaction which involves the formation of an e n z y m e - a m i n o a c y l adenylate complex with subsequent transfer of the amino acid to a molecule of t R N A specific for the amino acid (for reviews, see refs 1 and 2). Little is k n o w n of the structure of these enzymes or of which amino acid residues form the active site in the catalytic reaction. Since all of the synthetases catalyze the same t y p e of reaction, it seems possible that they do so by similar mechanisms. If this is true, then it is also possible that t h e y evolved from a c o m m o n ancestor. Analogy for such a relationship is seen with the mammalian serine proteases which catalyze similar reactions and are homologous [3]. Although these hypotheses can be tested b y sequencing the synthetases, such a task is formidable in view of the size of the proteins and the difficulties in obtaining them in large quantity. A second approach, a_,~d that which we have investigated, is to label amino acids at the catalytic sites using site-specific reagents with the intention of isolating and sequencing peptides containing the modified amino acid [4]. This approach has been used very successfully b y Shaw and coworkers [5,6] to label specific histidine residues in chymotrypsin and trypsin with tosyl phenylalanyl chloromethylketone and tosyl lysyl chloromethylketone, respectively. More recently, Powers and T u h y [7] have used peptidyl chloromethylketones to inactivate elastase. Bruton and Hartley [8] have labeled a lysine residue of N-formylmethionyl-tRNA synthetase using p-nitrophenylformylmethionyl-tRNA. However, this must be regarded as a special case, since other t R N A synthetases require a free amino group. Since accumulated evidence indicates that the carboxyl group of the substrate amino acid is not necessary for binding to the enzyme [ 9 - - 1 3 ] , we felt that reactive structural analogs modified at the carboxyl end would be recognized b y their respective enzymes and therefore might function as active sitespecific irreversible inhibitors. A reactive function at this position should be well situated for modification of at least one of the active site groups involved in proton transfer or substrate binding. For these reasons, we synthesized several amino acid chloromethylketones having the general structure, RO

Ill

X- H3N+-C-C-CH2C1

I

H

and studied their interaction with aminoacyl-tRNA synthetases.

79 Experimental

Halogen determination The Cl- and Br- content of the inhibitors was determined by potentiometric titration with silver nitrate, using silver and mercurous sulfate electrodes. Accuracy, -+ 5%. Enzyme assay Enzyme activity was measured by determining the rate of forming aminoacyl-tRNA [14,15]. The assay mixtures (0.1 ml) contained the following components: ATP (1.0 ~mole); MgC12 (1.5 umole); Tris buffer (pH 7.5, 7.5 ~mole); mercaptoethanol (1.4 pmole, except 1,0 pmole in the leucine assay}; tRNA (E. coli B, 0.2 mg); [3H]amino acid, 1.0 nmole; valine, 50--1000 Ci/mole; leucine, 100 Ci/mole; phenylalanine, 200 Ci/mole; lysine, 1000 Ci/mole; enzyme. Incubation at 37°C from two to six minutes gave a linear relationship between the product formed and the enzyme added. The reaction was terminated with 4 ml of 7% trichloroacetic acid. The precipitate was filtered on a Millipore filter, type HAWP, dried at 120°C for 20 min, and counted in a scintillation spectrometer. Enzyme preparations Experiments involving the effects of inhibitors on the aminoacyl tRNA synthetases were performed using a crude enzyme extract from frozen E. coli B cells harvested in late log phase (Grain Processing Corp.). Method 1 was nearly identical to that employed by Bergmann [16] in his preparation of fraction AS-1. Method 2 involved sonication of a bacterial cell suspension for 15 min, removal of nucleic acids by addition of MnCI2, and precipitation of protein with ammonium sulfate (75% saturation). Final centrifugation and dialysis were identical to Method 1. Synthesis of amino acid chloromethylketones The general procedure is illustrated by the synthesis of L-valyl chloromethylketone. The procedure for the synthesis of N-(p-nitrocarbobenzyloxy)-L-valine and the acid chloride was adopted from that of Carpenter and Gish [17]. L-Valine {5.86 g, 0.05 mole) was dissolved in 15.6 ml of 4 M NaOH and cooled in an ice-bath, p-Nitrocarbobenzyloxy chloride (13.5 g, 0.0625 mole) was dissolved in 32 ml of dioxane and the solution cooled in an ice-bath. The p-nitrocarbobenzyloxy chloride solution and 15.6 ml of cold 4 M NaOH were added to the valine solution in five approximately equal portions with a minimum of 5 min between additions. The mixture was stirred continuously at room temperature. After the last addition the mixture was stirred for an additional hour and then filtered. The filtrate was acidified with concentrated HC1, and the oil which separated was extracted into ethyl acetate using three 35-ml portions. The ethyl acetate phase was washed twice with 35-ml portions of 1 M HC1 and the product extracted into 1 M NaHCO3. The alkaline solution was washed twice with ethyl acetate, cooled to 0°C, and acidified with concentrated HCI. The product solidified upon standing in the cold; yield, 10.25 g (69%); m.p.,

80 74--75.7°C. A portion {8.88 g, 0.30 mole) of the product, N-(p-nitrocarbobenzyloxy)-L-valine, was dissolved in 170 ml of anhydrous ether and cooled in an ice-salt bath. Phosphorous pentachloride (7.0 g, 0.34 mole) was added and the mixture was stirred and cooled, with the exclusion of water. After one hour the mixture was allowed to warm to r o o m temperature and was then filtered through anhydrous sodium sulfate. The ether was evaporated and the resultant oil was washed three times with petroleum ether to remove POCI3. The acid chloride was dissolved in anhydrous ether and added with stirring to three equivalents of diazomethane in a cold ethereal solution. The solution was placed in the cold overnight. Thin layer chromatography on silica gel with ethyl ether revealed two spots {RF 0.56 and 0.80). Treatment of the ethereal solution with gaseous HC1 caused disappearance of the slower moving material and an increase in intensity of the faster moving material, identifying them as the diazomethylketone derivative and the chloromethylketone derivative, respectively. Thin-layer chromatography (ether--petroleum ether, 75:25, v/v) indicated two impurities (R F 0.43 and 0.91) in addition to the product (R F 0.71). The product was partially purified on a silica gel column usir_g ether--petroleum ether (75:25, v/v) as the eluant, and recrystallized from ether; yield, 46%; m.p. 72--73.5°C; (found: C, 51.4; H, 5.1; N. 8.5; C1, 11.1; C~4H, 7OsN2CI requires C, 51.2; H, 5.2; N, 8.5; C1, 10.8%). For removal of the blocking group, 329 mg (1.0 mmole) of the blocked chloromethylketone was dissolved in 10 ml of ethyl acetate and cooled in an ice-water bath. Gaseous HBr was bubbled into the solution for several minutes and the solution was allowed to stand at r o o m temperature with exclusion of moisture. The progress of the reaction was followed by thin layer chromatography (chloroform) where the starting material and the p-nitrobenzyl bromide had respective R F values of 0.36 and 0.85. After 4 h the solvent was evaporated, and the solid product was washed by centrifugation with ethyl acetate and anhydrous ether; yield, 100 mg (43%). Halogen titration indicated the presence of 1.96 halogen equivalents per mole with 8.2% of the product being in the form of the b r o m o m e t h y l k e t o n e . By shortening the reaction time (25 min}, with concommitant reduction in yield, a 16% yield of pure chloromethylketone was obtained; m.p., 142.5--144 ° C; pK a, 7.15 + 0.05; ultraviolet absorption Xrnax 278 nm (e 47); (Found: C, 31.5; H, 5.6; N, 6.4; C1, 15.5; Br, 34.2; C6 H, 3 NOC1Br requires C, 31.3; H, 5.7; N, 6.1; C1, 15.4; Br, 34.7). The chloromethylketone of D-valine was prepared in the same manner as the L-analog. Optical rotatory dispersion curves of the D- and L-compounds were opposite in sign and equal in magnitude: for L-valyl chloromethylketone in water; (c, 0.60), 23°C; [a]~00 0 °, [ a ] s s 9 0 °, [~]370 + 5.4 ° , [ ~ ] 3 s 0 + 8.8 ° , [~]330 + 1 9 ° , [ a ] 3 , 9 + 2 5 °, [ ~ ] 3 , 0 + 2 0 ° , [~]300 + 4 . 1 ° , [ ~ ] 2 9 s 0 °, F

[a]29o --16°, [a]28o --30 °, [~]27o --33 °, [0/]260 --31°, [~]2so --24°Inhibition studies Inhibition studies were performed using a crude e n z y m e extract diluted in 0.1 M potassium phosphate buffer at pH 7.3 containing 0.8 mg/ml bovine serum albumin. Unbuffered aqueous solutions of the inhibitors were prepared immediately before use, and appropriate aliquots were added to the diluted enzyme solutions. Inactivation was allowed to proceed at r o o m temperature

81 (23°C) and e n z y m e activity was measured by removing 25-pl aliquots and adding them to 75 pl of assay mixture containing 100 mM mercaptoethanol. (Mercaptoethanol, which reacts rapidly with the inhibitor, and dilution effectively terminate the inhibition reactions. Some error due to incomplete termination is observed at very rapid inactivation rates, but not under the conditions used in kinetic studies described here.) The valyl c h l o r o m e t h y l k e t o n e compounds used for the inactivation studies contained between 5 and 8 percent of the b r o m o m e t h y l k e t o n e , which had no noticeable effect on inactivation rates. All data were corrected for dilution factors and for normal enzyme inactivation under the experimental conditions. Kinetic constants for the process KI E+I ~ E'"I

k2 -~ El

(where K I is the dissociation constant of the dissociable enzyme-inhibitor complex, E-.'I, and k2 is the catalytic constant for formation of the covalent complex, EI) were determined by evaluating the rate constants for inactivation at room temperature as a function of inhibitor concentration. The rate constants (k0) were determined by measuring the fractional inactivation after one minute exposure to inhibitor with the assumption t h a t the decay enzyme activity follows a first order process [18] : In (Ea/Eo) = ko t. Experiments were performed to determine the effect of the various substrates on the inactivation of the valyl- and leucyl-tRNA synthetases by L-valyl c h l o r o m e t h y l k e t o n e and chloroacetone. After the addition of inhibitor to the e n z y m e solution containing the appropriate protecting agent, 25-tal aliquots of the enzyme solution were removed at appropriate intervals and added to 75/al of assay mixture containing 100 mM mercaptoethanol. Enzymatic activity was then determined. In cases where the protective agent was either the cognate amino acid or ATP + MgC12, or both, 1.0-ml samples of the inhibited e n z y m e were removed at appropriate intervals and added to 7 pl (100/Jmole} of mercaptoethanol. Before assay, the samples were dialyzed in the cold in 0.1 M potassium phosphate buffer at pH 7.3 containing 10 mM mercaptoethanol in order to remove the protective agent. Control samples not containing any protective agent but including inhibitor were subjected to the same procedure. Results

Synthesis of amino acid chloromethylketones The p-nitrocarbobenzyloxy amino acid chloromethylketones and the deblocked amino acid chloromethylketones which were synthesized are tabulated in Table I. Because some of the chlorine was displaced by bromide ions in the process of deblocking with HBr, mixtures of chloro- and b r o m o m e t h y l k e t o n e were produced; the b r o m o m e t h y l k e t o n e was the predominent product in the case of the glycine derivative. As indicated in the experimental section, we were able to synthesize small amounts of pure L-valyl chloromethylketone. The L-isoleucyl c h l o r o m e t h y l k e t o n e was obtained as an oil and therefore was n o t used in any inhibition studies.

82 TABLE I PHYSICAL CHARACTERISTICS

OF AMINO ACID CHLOROMETHYLKETONES

Chloromethylketone compound

m . p . (°C)

p - N O 2 C b z GIy Gly p - N O 2 C b z Lelle L-Ile b p-Cbz L-Phe L-Phe p - N O 2 Cbz L - V a l L-Val p - N O 2 Cbz D-Val

109=-110 78--79.5

Major infrared absorption b a n d s ( c m -1 )

% Bromomethylketone a

2 9 9 0 (NH3+), 1 7 2 5 ( C = O )

79

+ 2 9 5 0 ( N H 3 ), 1 7 2 0 ( C = O )

0

116.5--118 2 9 5 0 (NH3+), 1 7 1 5 ( C = O )

N.d. c

72--73.5 2 9 4 5 (NH3+), 1 7 3 5 ( C = O )

~

8

72--74

a F o r m e d by exchange d u r i n g r e m o v a l o f p - N O 2 C b z g r o u p b y HBr. b Oil. c N.d., not determined.

Stability of amino acid chloromethylketones The instability of the amino acid chloromethylketones became apparent upon their dissolution in water or buffer at pH 7.3. Glycyl chloromethylketone dissolved in water yielded a solution which became progressively more yellow in color. Phenylalanyl chloromethylketone y i e l d e d a clear aqueous solution, but when dissolved in pH 7.3 buffer formed a solution which immediately became turbid. Valyl chloromethylketone, when dissolved in buffer at pH 7.3, yielded a solution which became progressively turbid and yellow in color during the period of an hour. The instability of the amino acid chloromethylketones was reflected in a time~lependent change in their ultraviolet spectra. In the case of valyl chloromethylketone the band appearing at 327 nm reached one-half its maximum intensity in a b o u t 45 min. However, valyl chloromethylketone solutions which were exposed to pH 7.3 buffer for 40 min had considerably less than one-half the inhibitory activity of fresh solutions. Inactivation of aminoacyl-tRNA synthetases by L- and D- valyl chloromethylketones Fig. 1 shows that valyl chloromethylketone concentrations that rapidly inactivate the leucine and valine enzymes have little effect on the phenylalanine and lysine enzymes. The neutral inhibitor, chloroacetone, shows a similar pattern though much higher concentrations are required for comparable rates of inactivation {Fig. 2). In Fig. 3 it is seen that D-valyl chloromethylketone is nearly as effective as the L-analog inhibiting the leucine and valine enzymes. At high levels of inactivation, semilogarithmic plots of inactivation deviate from the expected straight lines {Figs 1--3). The primary cause of the nonlinearity at long times appears to be decomposition of the inhibitor, which reduces its concentration. Determination o f kinetic constants Dissociation and catalytic constants were evaluated according to the

83

(4.3mJ4)

~ 5o k) ~wZ

10.

~ •

(Jn~ ~I nLJ

"~ ~

\~

~

[VAL] (4raM)

[LEU](IX"

raM}

t MI NUTE5 Fig. 1. S e m i l o g a r i t h m i e p l o t o f t h e i n a c t i v a t i o n o f leucyl-, valyl-, p h e n y l a l a n y l - a n d l y s y l - t R N A s y n t h e t ases ( b r a c k e t s ) b y d i f f e r e n t c o n c e n t r a t i o n s ( p a r e n t h e s e s ) of L-valyl c h l o r o m e t h y l k e t o n e . The c u r v a t u r e o f t h e Hnes is d u e t o d e c o m p o s i t i o n of t h e i n h i b i t o r in the b u f f e r . The d a s h e d lines i n d i c a t e t h e p l o t s e x p e c t e d , a s s u m i n g n o d e c o m p o s i t i o n of t h e i n h i b i t o r .

1 0 0 ~ ~ ~ ; ~

~ --

Y ~(4.3mid)

NAU {4raM) p

[LIEU] ( h x )

~ NAt.](20raM) MINUTES Fig. 2. S e m f l o g a r i t h m i c p l o t of t h e i n a c t i v a t i o n o f leucyl-, valyl-, p b e n y l a l a n y l - a n d IysFI-tRNA s y n t h e t ases ( b r a c k e t s ) b y d i f f e r e n t c o n c e n t r a t i o n s ( p a r e n t h e s e s ) o f c h l o r o a c e t o n e .

100 5O h-

i

g

,2

l'e

20

MINUTES Fig. 3. S e m / ] o g a r i t ~ i e methylketone.

p l o t o f t h e i n a c t i v a t i o n o f leucyl- and v a ] y l - t R N A s y n t h e t a s e s by D-valyl c h l o r o -

T A B L E II

20116--23] 100185--115] c

1.811.5--2.0] 3.5[2.9--4.0] c

4 --16 7.5--45 32 - - 8 0

7.5 [ 6 . 9 - - 8 . 3 ] 4.5 [4.3--5.0] 103180 --135]

3.4[3.0--4.5] 4.0[3.6--4.3] 5.6[4.0--6.6]

k2 ( m i n - 1 ) a

1--8 1--8 8--64

Inhibitor c o n c n (raM) b

a L e a s t s q u a r e s v a l u e s for K I a n d k 2 are given; t h e v a l u e s in b r a c k e t s i n d i c a t e r a n g e s d u e t o e x p e r i m e n t a l e r r o r . b R a n g e of i n h i b i t o r c o n c e n t r a t i o n s used; a t l e a s t f o u r c o n c e n t r a t i o n s w e r e u s e d f o r e a c h d e t e r m i n a t i o n a n d e a c h d e t e r m i n a t i o n w a s r e p e a t e d at ]east t w i c e . c Did n o t e x h i b i t s a t u r a t i o n kinetics.

L-Valyl c h i o r o m e t h y l k e t o n e D-Valyl c h l o r o m e t h y l k e t o n e Chloroaeetone

KI(mM) a

Inhibitor concn (mM) b

KI(mM) a

k 2 ( m i ~ - 1 )a

Leucyl-tRNA synthetase

INHIBITORS

Valyl-tRNA synthetase

KINETIC C O N S T A N T S OF C H L O R O M E T H Y L K E T O N E

Ov

85

1

~

60

0

0

~

I

~ 2o

MINUTES Fig. 4. I n h i b i t i o n o f v a l y l - t R N A s y n t h e t a s e b y 8 m M L - v a l y l c h l o r o m e t h y l k e t o n e (o), a n d in t h e p r e s e n c e o f e i t h e r 2.8 m M I.fvaline (A), 28 m M A T P + 7 0 m M MgCI 2 ( i ) , or b o t h ( o ) . T h e lines j o i n i n g t h e p o i n t s are i n t e n d e d o n l y to i n d i c a t e trends.

method of Schaeffer et al. [18] using Lineweaver--Burk double reciprocal plots of l/k0 versus 1/I. The kinetic constants are listed in Table II. The m e t h o d employed for evaluation of the first order rate constant (k0) is only valid if the extent of inactivation is within the linear portion of the first order rate plot. As seen in Figs 1--3, the plots do deviate from linearity. However, the deviation during the short times (1 min) used in the k0 determinations was so small (within experimental error} that corrections were unnecessary. Therefore the data in Table II are uncorrected.

Substrate protection Cognate amino acid and/or ATP + Mg 2+ protected the valyl- and leucylt R N A synthetases from inhibition b y L-valyl chloromethylketone (Figs 4 and 5}, while other amino acids or t R N A failed to afford protection (Figs 6 and 7). A similar pattern of protection was exhibited when chloroacetone was used as the inhibitor. Considerable scatter of points was observed in the experiments depicted in Figs 4 and 5 because of the necessity of dialyzing each sample to remove protecting amino acids before assay. However, the ability, or lack of it, of various substrates to protect the enzymes is readily apparent b y comparing the amount of enzyme activity remaining after the 8-min incubation time with inhibitor (cf. Figs 4 and 6). 100

~u 80 ~z 60 40

20

2

4

6 MINUTES

8

Fig. 5. I n h i b i t i o n o f l e u c y l - t R N A s y n t h e t a s e b y 1 m M L-valyl c h l o r o m e t h y l k e t o n e ( e ) , and in the prese n c e of e i t h e r 2.4 m M L - l e u c i n e (A), 1 4 . 5 m M A T P + 36 m M MgCI 2 (m), or b o t h (o).

86 1001 C--- 8 0

(J < F-

Z uJ

60

~ 4o W

20

MINUTES Fig. 6. I n h i b i t i o n o f v a l y l - t R N A s y n t h e t a s e b y 8 m M L-valyl e h l o r o m e t h y l k e t o n e (e), and in t h e p r e s e n c e o f e i t h e r 2 . 8 m M L - l e u c i n e (A), 2 . 8 m M L - p h e n y l a l a n i n e (m), o r 5 . 0 m g / m l t R N A (o).

Discussion

Synthesis and properties of amino acid chloromethylketones Amino acid chloromethylketones were synthesized by essentially the same procedures used by Shaw et al. [5] for the synthesis of tosyl lysyl and phenylalanyl chloromethylketones. The p-nitrocarbobenzyloxy group was used to block the amino group during subsequent synthetic steps when it was found that attempts to convert carbobenzyloxy amino acids to acid chlorides generally led to the formation of N-carboxy anhydrides [19,20]. The amino acid chloromethylketones are unstable under conditions used for inactivation studies, i.e., in pH 7 buffer. The mode of decomposition is unknown. The compounds are relatively stable as salts in water, but decompose when the pH of solutions is raised to values near the apparent pK a (ca. 7.2) of the amino group, as seen by the time-dependent appearance of a new ultraviolet-absorbing peak at 327 nm and a decrease in inhibitory ability. The 327-nm band may be due to a secondary decomposition product, since none of the expected products (see below) would absorb at this wavelength. Furthermore it can be calculated, assuming that the deviation from linearity of the inactivation curves (cf. Fig. 1) is due to decomposition, that the half-life for decomposition

>-

~00

<

~z 60 13_ 2C

2

4

6

8

MINUTES Fig. 7. I n h i b i t i o n o f l e u c y l - t R N A s y n t h e t a s e b y I m M L-vzdy] c h i o r o m e t h y l k e t o n e ( o ) , a n d i n t h e prese n c e o f e i t h e r 2 . 4 m M L-valLne (A), 2 . 4 m M L - p h e n y l a l a n i n e (m), o r 1 0 . 9 m g / r n / t R N A (o).

87 is a b o u t 10 min, compared to a half-life of 45 min for appearance of the 327-nm band. Decomposition cannot be explained by simple hydrolysis of the chloromethyl group, since chloroacetone is relatively stable under the same conditions. The side chain also has an influence on stability, valine chloromethylketone being much more stable than the other inhibitors studied. This effect may be explained by the "rule of six" [21] according to which the side chains of valine and isoleucine should provide more steric hindrance to reactions involving the amino and carbonyl groups. Steric hindrance is also reflected in the degree of displacement of chloride b y bromide ions {Table I) during HBr deblocking of the p-nitrocarbobenzyloxy derivatives. The available data suggest that decomposition of amino acid chloromethylketones involves a reaction of the u n p r o t o n a t e d amino group, e.g. intramolecular general base-catalyzed hydrolysis of the chloromethyl by the amino group, or intermolecular attack on a carbonyl to form a Schiff-base dimer. The instability of the chloromethylketones may limit this approach to the study of only a few aminoacyl-tRNA synthetases (valyl-, leucyl-, isoleucyl- and perhaps prolyl-). A knowledge of the mode of decomposition would be helpful in designing other types of inhibitors which are more stable.

Site of reaction of amino acid chloromethylhetones Several lines of evidence suggest that L-valyl c h l o r o m e t h y l k e t o n e inhibits by reaction at the active sites of the valyl and leucyl-tRNA synthetases. 1. The L- and D-valyl chloromethylketones exhibit saturation kinetics with respect to the inactivation of the valyl- and leucyl-tRNA synthetases. This is characteristic of active site-specific reagents [18]. Their dissociation constants {Table II) are similar in magnitude to those of other competitive inhibitors [9,10]. Saturation kinetics with respect to the leucyl enzyme was unexpected but is supported by the report of R o u g e t and Chapeville [22] indicating the existence of an interconvertible form of leucyl-tRNA synthetase which catalyzes valine dependent pyrophosphate exchange. It should be n o t e d that at pH 7.3, only a b o u t 40% of the valyl chloromethylketone (pK a = 7.15) exists in the protonated form, while nearly 100% of the natural substrate, L-valine (pK a = 9) is protonated. If, as seems likely, the enzyme requires the pro~onated form for efficient binding, then the experimentally determined K I values must be decreased by a factor of 2.5 to arrive at the actual dissociation constants. 2. The L- and D-valyl chloromethylketones exhibit differences with respect to their dissociation constants from the valyl- and leucyl-tRNA synthetases. This indicates that the valyl-tRNA synthetase discriminates between the L- and D-isomers, as would be expected for an active-site inhibitor and a stereospecific enzyme. The catalytic constant (k2 = 3.4 min -~ ) of D-valyl chlor o m e t h y l k e t o n e with the valyl synthetase is larger than that of the L-analog (h: = 1.8 min -~), indicating that although the D-compound binds five times less effectively to the enzyme, the chloromethylketone group is more ideally positioned for reaction at the active site. In contrast to L - and D-valine, which show a 50-fold difference with respect to their dissociation constants from valylt R N A synthetase [ 9 ] , the L- and D-valyl chloromethylketones show only a 5-fold difference. This is consistent with the data of Owens and Bell [9] which

88 indicate that the L - and D-forms of competitive inhibitors of valyl-tRNA synthetase show much smaller K I differences than the L - and D-forms of valine. It is also interesting to note that the catalytic constant (k2) of L-valyl chloromethylketone with valyl-tRNA synthetase is approximately one-half as large as the constants for the other inactivations. 3. The valyl- and leucyl-tRNA synthetases are protected from inactivation only by their natural substrates. This is a criterion required of reagents acting as active site-specific inhibitors, but is n o t proof in itself since protection was apparent even when the non-specific reagent chloroacetone was used as inhibitor. Protection in the presence of ATP + Mg 2÷ is indicative of "burial" of that portion of the enzyme which reacts with the inhibitor. Valine does not protect the leucyl-tRNA synthetase {Fig. 7) although L- and D-valyl chloromethylketone appear to bind to it. A possible explanation is that the carboxyl group of valine somehow prevents efficient binding of valine to the leucyl-tRNA synthetase. Accordingly L-leucine, which does not protect the valyl enzyme (Fig. 6), binds 400 times less efficiently to the valyl enzyme than does L-valine [9]. It is interesting to note {Fig. 7) that t R N A appears to promote inhibition of the leucyl enzyme. This effect, although n o t observed with the valyl enzyme, was reproducible and suggests a possible conformational change resulting in increased exposure of a portion of the active site. These experiments seem to indicate that the amino acid modified is involved in binding or proton removal from ATP or the substrate amino acid, but n o t t R N A , since the former protect the enzymes from inactivation, while the latter does not. 4. The pattern of inactivation of the aminoacyl-tRNA synthetases differs significantly from that observed in the presence of the sulfydryl reagent pchloromercuribenzoate. Stern et al. [ 2 3 ] , using 1 mM p-chloromercuribenzoate, observed losses in the enzymatic activities of the leucyl-, valyl, and phenylalanyl-tRNA synthetases of 100%, 90% and 100%, respectively. The activity of lysyl-tRNA synthetase was found to increase by 50%. One would expect a similar pattern of inactivation if L-valyl chloromethylketone were reacting simply with the sulfhydryl groups of the synthetases. These experiments, however, do not exclude the possibility of reaction with non-functional sulfhydryls. 5. Chloroacetone, which bears little structural resemblance to valine, does not exhibit saturation kinetics with respect to valyl-tRNA synthetase. In fact, the first order rate constant increases at a faster rate than the increase in chloroacetone concentration. While this reagent may inhibit b y reaction at the same site as valyl chloromethylketone, it is apparent that the reaction is complex, probably because of non-specific alkylation at other sites, also. In retrospect it is perhaps not surprising that the amino acid chloromethylketones do not display an absolute specificity for their target t R N A synthetases, since several of them, the isoleucyl and valyl enzymes in particular, have only a limited ability to discriminate between related amino acids, as measured in the ATP:PP i exchange reaction [ 2 ] . Under physiological conditions, however, the enzyme is almost entirely complexed with its cognate t R N A [ 2 4 ] , which plays a key role in determining the specificity of the enzyme [2,25]. Therefore it may be fruitful to study the inhibition reactions in more detail with purified enzyme and tRNA. In a recent communication Frolova et al. [ 2 6 ] , described the inactivation

89 of purified beef liver valyl-tRNA synthetase b y valyl chloromethylketone. Their results (observation of saturation kinetics and protection b y valine) are in substantial agreement with ours, except that their kinetic constants (K I = 0.7 mM; k2 = 0.02 min -' ) are quite different. This may, however, be due to species difference such as we have seen in the inactivation of yeast and pig heart fumarase b y an irreversible inhibitor [ 2 7 ] . The surprising result from the work of Frolova et al., is the apparent stability of their inhibitor, since their studies were carried o u t at concentrations where we observed very little inhibition, because of decomposition. This descrepancy remains to be resolved. The isoleucyl, leucyl and valyl synthetases of E. coli resemble one another in several respects, including molecular weight, state of aggregation (monomer), and sensitivity to antisulflydryl agents [ 2 ] , as well as structure of substrate amino acid. In view of these similarities and possible evolutionary relationships, this group of enzymes would seem to be the logical target of future studies with the valyl and isoleucyl chloromethylketones.

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