[21] N-substituted arginine chloromethyl ketones

[21]

N-SUBSTITUTEDARG1NINE CHLOROMETHYLKETONES

229

primarily in a catalytic role in the carboxypeptidase-catalyzed hydrolysis of peptide substrates. Bromoacetyl-p-aminobenzyl succinic acid alkylates a methionyl residue in carboxypeptidase B2 As shown above, similar reagents modify amino acids other than methionyl residues, i.e., Tyr-248 and Glu-270. In those cases, the alkylating moiety was located in close proximity to the scissile bond of a normal substrate. Therefore, it would most likely interact with a residue that presumably functions in the catalytic step. The hydrophobic nature of bromoacetyl-p-aminobenzylsuccinate and the fact that the reactive side chain is carried on the aromatic part of the molecule suggests that the methionyl residue modified is part of the substrate recognition site in the hydrophobic pocket of carboxypeptidase B. ~,9,~ This possibility is reminiscent of the role of Met-192 in chymotrypsin, where it appears to function as a flexible hydrophobic lid on the substrate binding pocket. ~°R. G. Reeck, K. A. Walsh, M. A. Hermodson, and H. Neurath, Proc. Natl. Acad. Sci. U.S.A. 68, 1226 (1971).

[21] N - S u b s t i t u t e d

Arginine Chloromethyl Ketones B y B. KEIL

Although the early efforts to extend the chloromethyl ketones series to arginine derivatives met with chemical difficulties, two active sitedirected inhibitors of trypsin and trypsinlike enzymes are now available: N~-p-nitrobenzyloxycarbonyl-L-arginine chloromethyl ketone (p-NO2ZACK, Fig. la) ~ and N~-tosyl-L-arginine chloromethyl ketone {TACK, Fig. lb). 1 p - N O . , - Z A C K is a highly efficient inhibitor of trypsin ~-~ and an extremely rapid inactivator of clostripain. ~ T A C K is about three times more potent than T L C K ~ for the inactivation of trypsin, and it was found to be an efficient inhibitor of a trypsinlike enzyme from S t r e p t o m y c e s erythreus.'-'

~Abbreviations used: TACK, N"-tosyl-L-arginine chloromethyl ketone (L-l-ehloro3-tosylamido-6-guanidinohexan-2-one) ; p-NO...-ZACK, N'*-p-nitrobenzyloxycarbonyl-L-arginine chloromethyl ketone; TLCK, N~-tosyl-L-lysine chloromethyl ketone ; Tos- tosyl; Z-, benzyloxycarbonyl- ; DTT, dithiothreitol ; TLME, N~-tosylL-lysine methyl ester; TAME, N~-tosyl-L-arginine methyl ester. -~N. Yoshida, A. Sasaki, and K. Inouye, Biochim. Biophys. Acta 321, 615 (1973). :JE. Shaw and G. Glover, Arch. Biochem. Biophys. 139, 298 (1970). 4O. Siffert, I. EmSd, and B. Keil, FEBS Lett. 66, 114 (1976).

230

ENZYMES, ANTIBODIES, AND OTHER PROTEINS

[21]

/CO--CH2CI x)-----CH2O-- CO--NH--CH + ~(CH2 )a-- NH~ C = NH-

NO2----(z

I

NH2

~ H3C ~

'

~

/CO--CH2C] SO2-'--NH-- C.H,.

+

(CH2)3--NH--C~NH2

J

NH2

/CO~CH2

No~

c.~ o - c o - N. - c ~ (c.~) N - f

©

÷

cl= N.~ NH2

Fla. 1. Derivatives of arginine ehloromethyl ketone: (a) p-N0~-ZACK; (b) TACK ; (e) inactive eyelie derivative of p-N02-ZACK.

Method of Preparation of p-NO2-ZACK Shaw and GloveV made the first attempt to synthesize an arginine chloromethyl ketone derivative for affinity labeling of the active sites of trypsinlike enzymes. Although the synthesis yielded a mixture containing less than 2% of the active chloromethyl ketone (Fig. la) and mainly an inactive cyclic product (Fig. lc), they were able to show that this agent was a rapid inhibitor of trypsin. By a modification of the synthesis and the introduction of additional purification steps Siffert e t al. 4 prepared p-NO2-ZACK in a pure form. Their procedure is described here. p-NOr-Z-Arg-OH

PCls CHIN2 HCI > p-NO~-Z-Arg-C1 ~ p-NO~-Z-Arg-CHN~ ) p-NOrZ-Arg-CH~C1

~- (p-Nitrobenzyloxycarbonyl) arginine (706 mg) prepared according to Gish and Carpenter 5 is suspended in anhydrous tetrahydrofuran (25 ml), chilled in an ice-salt bath, and treated by adding phosphorus pentachloride (840 mg) in several portions with stirring. The mixture is stirred for 120 min during which time the temperature is allowed to rise to 0% After addition of anhydrous ether (100 ml) the precipitate is 5D. T. Gish and F. I-I. Carpenter, J. A m . Chem. Soc. 75, 5872 (1953).

[21]

N-SUBSTITUTED

ARGININE

CHLOROMETHYL

231

KETONES

filtered, washed with ether, and dried under reduced pressure over sodium hydroxide pellets at 4 °. The yield of resulting a-(p-nitrobenzyloxycarbonyl)arginyl chloride hydrochloride is 700 mg (86%); m.p. = 120°; infrared spectrum (KBr) = 1785 cm-1. To the product dissolved in anhydrous tetrahydrofuran (15 ml) is added at 0 ° an excess of ethereal solution of diazomethane, until a yellow color persists. After an hour of stirring, 80 ml of anhydrous ether are added. The light yellow precipitate is filtered, washed several times with anhydrous ether, and dried under reduced pressure. The yield is 500 mg (77%) of the diazo derivative. To 380 mg (1 mmole) of this product is added 1 N HC1 in acetic acid (15.2 ml) at room temperature. After an hour, evolution of nitrogen ceases. The solvent is evaporated under reduced pressure at 35 ° , the last traces being removed with an oil pump. The resulting crude p-NO~-ZACK is purified consecutively by two procedures: after passage through a column of silica gel (Kieselgel, methanol-chloroform 9:1 v/v) the product is taken to dryness, dissolved in water, and freed from salts by passage through a column of Bio-Gel P-2 (400 mesh). Lyophilization of the eluate gives 150 mg of the desired p-NO2-ZACK, m.p. 98-105 ° (38% yield). Thin-layer chromatography (Kieselgel GE 254, Merck; 1-butanol-acetic acid-water 18:2:5 by volume) followed by the Sakaguchi reaction reveals a single spot. Infrared spectrum (KBr) = 1739, 1700, 1675, 1520 cm-1. A 14C-labeled reagent can be synthesized by this procedure using labeled diazomethane for the conversion of a-(p-nitrobenzyloxycarbonyl) arginyl chloride to the diazoderivative2 Method of Preparation of TACK Inouye e t al. ~ developed two syntheses for TACK, starting with either NG-nitro - or Na-tosylarginine. The first variant, yielding pure and stable products, will be described here. H-Arg(NO2)-OH

Tos-C1

PCls

~ Tos-Arg(NO2)-OH ~

Tos-Arg(NO2)-CHN2

HF

Tos-Arg(NO2)-C1

* Tos-Arg(NO.o)-CH2C1

CH2N,

,

HC1

~Tos-Arg-CH2C1

To a solution of NG-nitroarginine (5.5 g) in 2 N NaOH (37.5 ml) is added sodium carbonate (2.65 g) and acetone (25 ml) followed by a dropwise addition of p-toluenesulfonyl chloride (7.15 g) in acetone 60. Siffert, in preparation. K. Inouye, A. Sasaki, and N. Yoshida, Bull. Chem. Soc. Jpn. 47, 202 (1974).

232

ENZYMES, ANTIBODIES, AND OTHER PROTEINS

[21]

(20 ml) at 0% After stirring at 0 ° for 3.5 hr and subsequent removal of acetone under reduced pressure, the aqueous solution is acidified with 4 N HC1. The crystalline precipitate is filtered, washed with water, and dried under reduced pressure. Pure tosylnitroarginine is obtained after recrystallization from methanol-water (7.39 g, 79%), m.p. 167°-1697C. To the chilled solution of 750 mg of tosylnitroarginine in anhydrous tetrahydrofuran (10 ml) in an ice-salt bath is added phosphorus pentachloride (840 mg) in several portions. The temperature is allowed to rise to 0 ° during an hour of stirring. Addition of 80 ml of ahhydrous ether gives a syrupy precipitate, which, after decantation of the supernatant fluid and trituration with ether, is dried under reduced pressure over sodium hydroxide pellets at 4 °. The resulting amorphous tosylnitroarginyl chloride (470 rag, 60%) is dissolved in anhydrous tetrahydrofuran (10 ml) and an excess of an ethereal solution of diazomethane is added at 0 ° until the yellow color persists. After 1 hr of stirring at 0 °, the diazo derivative is precipitated by addition of anhydrous ether (80 ml), filtered, washed with ether, and dried under reduced pressure (360 mg, 45%). To 250 mg of the diazo derivative is added 1 N HC1 in acetic acid (10 ml) at room temperature. After an hour the evolution of nitrogen ceases. The solvent is evaporated under reduced pressure at 35 ° and, after addition of acetic acid, the product is lyophilized. Crude tosylnitroarginine chloromethyl ketone is purified by passage through a column of silica gel (25 g, mesh 0.05-0.2 mm; Merck) in methanol-chloroform (1:9 by volume). Fractions containing the desired product (as followed by thin-layer chromatography) give, after evaporation of the solvent, a syrup that crystallizes upon addition of Chloroform (120 mg, 49%). Pure nitroderivative is obtained after recrystallization from methanol (86% yield). This intermediate, 180 mg, and 0.2 ml of anisole are placed in a reaction vessel made of fluorinated polyethylene and, after chilling the mixture in a Dry Ice-acetone bath, about 5 ml of hydrogen fluoride are introduced. The mixture is stirred at 0 ° for 30 min, and hydrogen fluoride is evaporated under reduced pressure. The residue is dissolved in water, washed with ethyl acetate, and passed through a column (0.9 X 10 cm) of Amberlite CG-400 in acetate form with subsequent elution by water. To the combined eluates is added 0.5 ml of 1 N HCI; subsequent lyophilization yields 180 mg of the desired N~-tosylarginine ehloromethyl ketone (98%). Infrared spectrum (KBr) = 1737 cm-1, [~]~5 = _21.2 ° ± 0.6 ° (c 1.0, 0.1 N HC1). Thin-layer chromatography (cellulose F, Merck) in 1-butanol-acetic acid-water (18: 2: 5 v/v/v) followed by the Sakaguchi reaction reveals a single spot.

[21]

N-SUBSTITUTED ARGININE CHLOROMETHYL KETONES

233

Affinity Labeling of Trypsin and Trypsinlike Enzymes Inhibition o] fl-Trypsin by p-NO.,_-ZACK and TACK In a preliminary study on the inactivation of fl-trypsin by a preparation of 2% p-NO~-ZACK and 98% of the inactive cyclic compound (Fig. lc), Shaw and Glover 3 were able to show the high efficiency of the inhibitor. They estimated that pure p-NO~-ZACK should be orders of magnitude more rapid than the lysine derivative, TLCK. From their data it could I)e deduced that the impure inhibitor preparation totally eliminates the activity of trypsin within 10 min at pH 7 and 25 ° at an inhibitor-enzyme ratio of 50:1. They have also shown that a histidine residue is replaced by a 3-carboxymethylhistidine residue in the total hydrolyzate of the inactivated trypsin, which proves the active siteoriented action of the inhibitor. Siffert et al2 studied the inhibition of fl-trypsin by pure p-NO2-ZACK. The enzyme concentration was 10 ~M, inhibitor concentrations varied between 6 and 2.5 }( l0 -~ = 0.25 raM, and the pH was maintained at 7.0 at 25% Within 10 min, activity was completely lost at an inhibitor-enzyme ratio of 17:1. The study of the saturation kinetics of inactivation showed that the line could be drawn through the origin, indicating that essentially no reversible intermediary complexes were formed. Yoshida et al? studied the trypsin-TACK system and found values of Ki = 9 mM and K3 = 0.2 rain -1, i.e., TACK is 3.4 times more rapid than TLCK in inactivating trypsin. On the other hand, it is much less effective than p-NO2-ZACK. The authors 2,3 agree, however, that the increased effectiveness of p-NO._,-ZACK over TLCK may be due, in part, to the difference of side chains (Tos- and p-NO.,-Z-), not only to the advantage of arginine over lysine. The same effect has been observed in the case of inhibition of chymotrypsin and subtilisin by the phenylalanine chloromethyl ketone derivative, s,9

Inhibition o] the Serine Protease ]rom Streptomyces erythreus by TACK Streptomyces erythreus serine protease has a substrate specificity similar to that of bovine trypsin. A comparative study of its inactivation by TACK and TLCK made by Yoshida et al. 2 has shown that the rates of inactivation of the protease by the two inhibitors are not different, in contrast with the higher efficiency of TACK toward trypsin (see the table). 8 E. Shaw and J. Ruscica, J. Biol. Chem. 243, 6312 (1968). E. Shaw and J. Ruscica, Arch. Biochem. Biophys. 145, 484 (1971).

234

ENZYMES, ANTIBODIES, AND OTHER PROTEINS

[21]

KINETIC DATA FOR THE SUBSTRATES AND IRREVERSIBLE INHIBITORS OF

Streptomyces erythreus PROTEASE AND TRYPSIN a Inhibitor Substrate Kin. 10 -4

Ki • 10 -4 M

Enzyme

TLME

TAME

TLCK

TACK

S. erythreus protease

0.6 1.0

0.2 0.1

0.7 2.5

0.5 0.9

Trypsin

ks • min -1 TLCK TACK 0.14 0.16

0.11 0.22

a F r o m N. Yoshida, A. Sasaki, a n d K. Inouye, Biochim. Biophys. Acta 321, 615 (1973).

Inhibition of Clostripain by p-NO2-ZACK

Clostripain (EC 3.4.22.8) from the culture filtrate of Clostridium histolyticum is a thiol proteinase with a highly limited specificity directed at the carboxyl bond of arginyl residues in proteins and in synthetic substrates. Porter et al. 1° have described the inactivation of clostripain by TLCK. Their enzyme preparation (specific activity 1.1 ~kat/mg) incorporated approximately 4 moles of TLCK per mole of enzyme of molecular weight 50,000 instead of the expected 1:1 molar ratio. Because they found by independent means that the enzyme was homogeneous, they concluded that a large fraction of the enzyme was in an inactive state. Recently Siffert et al. 4 obtained complete inactivation of clostripain by pure p-NO2-ZACK. The enzyme they used was four times more active (4.5 ~kat/mg) than that of Porter. It was activated for 2 hr at room temperature prior to the assay at a concentration of 20 ~M in 50 mM Tris chloride at pH 7.4, containing 2.5 mM D T T and 50 mM CaCI~. In inhibition experiments at an initial enzyme concentration of 10 ~M, the rate of inactivation is extremely rapid: a 4M excess of the reagent removes the activity completely in less than 2 min at room temperature. Evidence that the reaction is oriented in both cases to the active site of clostripain is given by the results of experiments effected in the presence of a competitive inhibitor. Benzamidine (Ki = 42.9 mM) is effective in protecting the active site of clostripain from reaction with both TLCK 1° and p-NO2-ZACK. 4 At present, there is no direct experimental evidence as to which residue in clostripain is substituted by the action of the chloromethyl ketones. ~oW. H. Porter, L. W. Cunningham, and W. M. Mitchell, J. Biol. Chem. 246, 7675 (1971).

]22]

RENIN

235

Another question that remains open is the number of inhibitor residues incorporated in the enzyme. Although Siffert et al. 4 used a different reagent from that of Porter et al., 1° and a highly active enzyme, the resul~ts of the two studies indicate a molar ratio of 4 moles of the chloromethyl ketone per mole of enzyme.

Evaluation Enzymes cleaving polypeptide substrates at the carboxyl group of arginine residues can be irreversibly inhibited by two arginine chloromethyl ketones, p-N02-ZACK and TACK. The reaction is specifically oriented to their active sites. Both inhibitors can be prepared in pure and stable form. In all cases studied, p-NO~-ZACK was found more efficient than TACK. The highest rate of inactivation was observed with clostripain.

[22] Renin B y TADASHI INAGAMI and YASUNOBU SUKETA

Renin (EC 3.4.99.19), which plays a key role in renovascular hypertension and blood pressure regulation by producing angiotensin I, is a protease with an extremely limited substrate specificity: it will cleave only the unique leucylleucine peptide bond of angiotensinogen and the tetradecapeptide renin substrate H-Asp-Arg-Val-Tyr-Ile-His-Pro-PheH i s - L e u - L e u - V a l - T y r - S e r - O H . 1 This unique substrate specificity is reflected in stringent structural requirements for an extensive segment of substrate molecules. 2 Results of competitive inhibition experiments using various peptides possessing partial structures of the substrate indicate that only the Cterminal portion, Leu-Leu-Val-Tyr, of the tetradecapeptide substrate contributes significantly to the binding affinity of the substrate to the enzyme2,4 a-Bromoisocaproic acid has a hydrocarbon structure identical with that of leucine, its a-bromine atom substituting for the a-amino group ' L. T. Skeggs, J. R. Kahn, K. E. Lentz, and N. P. Shumw~y, J. Exp. Med. 106, 439 (1957). L. T. Skeggs, K. E. Lentz, J. R. Kahn, and H. Hochstrasser, J. Exp. Med. 128, 13 (1968). K. Poulsen, J. R. Burton, and E. Haber, Biochemistry 14, 3892 (1975). 4T. Kokubu, E. Ueda, S. Fujimoto, K. Hiwada, A. Kato, H. Akutsu, and Y. Yamamura, Nature (London) 217, 456 (1968).