/ . Biochem. 82, 645-651 (1977)

Studies on Papain Catalysis with Substrates Containing the 7>an5-/?-Phenylazobenzoyl Group as a Probe for Hydrophobic Interaction Motonori OHNO, Jun-ichiro SARUWATARI, and Masako WATANABE Laboratory of Enzyme Chemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, March 30, 1977

The susceptible bonds of substrates of the type PABz-Gly.-Arg-R (R=OCH3> n=0,l,2; R=OH, 71 = 1,2) to papain [EC 3.4.22.2] have been identified. PABz-Arg-OMe was found to be approximately 30 times more reactive than benzoylarginine ethyl ester, due to a XmCapp) value 25 times smaller. The finding that PABz-Glyi-Arg-OMe was specifically hydrolyzed at the ester bond with a second-order rate constant comparable with that of PABz-Arg-OMe indicated that the S4 subsite of the enzyme is capable of accommodating a large hydrophobic group. Other substrates were cleaved at the peptide bond one residue removed from the PABz group, in accord with the well-known fact that the S, subsite interacts preferentially with a hydrophobic P, residue. A negative charge on the substrate at the P,' or P,' position greatly decreases the acylation rate and also the noncovalent binding affinity. The hydrolysis of PABz-Arg-OMe and PABz-Gly,-Arg-OMe was little affected by the acridine dye proflavine.

The specificity of papain [EC 3.4.22.2] has been extensively studied using a variety of synthetic substrates. Schechter and Berger (/) showed that papain has an active site extending over approximately 25 A, which can be divided into seven subsites, each accommodating one amino acid residue of the substrate. Four of these subsites (Sj-SJ are located on the N-terminal side of the cleaved bond 1

All amino acids described in this paper are of L-configuration, except for glycine. Abbreviations: PABz, rron^-p-phenylazobenzoyl; OMe, methoxy; 2, benzyloxycarbonyl; BAEE, benzoylarginine ethyl ester; DTT, dithiothreitol; MeOH, methanol; EtOH, ethanol; DMSO, dimethyl sulfoxide; AcOH, acetic acid; tic, thin-layer chromatography; pc, paper chromatography. Vol. 82, No. 3, 1977

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and the other three (SZ-SJO on the C-terminal side. They further found that the S, subsite interacts preferentially with the side chain of hydrophobic amino acid residues such as Phe, Val, and Leu1 (2). Alecio et al. (3) showed with a series of substrates of the type benzyloxycarbonyKZ^-Gly-X-NHt, where X denotes various amino acids, that the Si' subsite interacts effectively with hydrophobic residues, in particular Leu and Trp. Lowbridge and Fruton (4) and Mattis and Fruton (5) showed with fluorescent substrates, 6-(N-methylanilino> naphthalene-2-sulfonyl-Gly.-Val-Glu-Leu-Gly (/»= 1, 2), in which the Glu-Leu bond is the only one cleaved, that secondary enzyme-substrate interactions are of importance in the efficiency of papain catalysis.

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M. OHNO, J. SARUWATARI, and M. WATANABE

In the present investigation, attempts have been a homogeneous solution. After cooling to 40°C, made to examine properties of the extended active the solution was acidified to pH 2. The precipitate site and catalytic features of papain with substrates was filtered off and washed with water (13 g, 90%). of the type /ra/iy-/>-phenylazobenzoyl(PABz)-Gly.- The product was recrystallized from methanolArg-R (R=OCH 3 , n=0, 1, 2; R=OH, n = \, 2). (MeOH)-ethyl acetate-1-hexane, 12.2 g (84%); cis- and /ra/w-PABz-Arg-OMe and their hydrox- mp 214-217°C; Rf^ (a) 0.91. Anal. Calcd. for amides were first introduced by Wainberg and Q J H ^ O J N , : C, 63.59; H, 4.63; N, 14.83. Found: Erlanger (6) as substrates for trypsin to explore a C, 62.89; H, 4.59; N, 14.59. hydrophobic slit at the active site. The p-phenylPABz-Glyt (II)—This was prepared from azobenzoyl group can exist as cis and trans isomers PABz-Q and Glyj in the manner described above. that are interconvertible under the influence of light Yield 94%; mp 237-238°C decomp.; Rf^ (a) 0.83. of certain wavelengths (7, 4N«: C, 59.99; H, 4.74; we used trans-PABz, which is stable under natural N, 16.46. Found: C, 59.73; H, 4.73; N, 16.36. light. In the trans configuration, the two benzene PABz-Arg-OMe-HCl (III)—This was prerings and azo nitrogens are in the same plane (9). pared from PABz-Cl and Arg-OMe-2HCl by the Thus it is of interest to compare the reactivities of method of Wainberg and Erlanger (6) with some PABz-Arg-OMe and benzoylarginine ethyl ester modification; chloroform and MgO were replaced (BAEE) toward papain, since the latter is commonly by dioxane and NaHCO, to form a homogeneous used as a specific substrate for this enzyme. solution. The product, obtained in 60% yield The susceptible bonds of the substrates to after recrystallization, melted at 203-205°C after papain were identified and estimates were then softening at 93-95°C; [a]D-16.4°C (c 2.0, MeOH); made of apparent Michaelis constant, Km(apP), and -R/Pc (a) 0.93. Anal. Calcd. for QoHuOaN,catalytic constant, Arc»t, in the hydrolysis of these 1/2H.O; C, 54.35; H, 5.93; N, 19.02. Found: substrates. The kinetics for ester substrates were C, 54.66; H, 5.80; N, 19.11. also determined in the presence of the acridine dye PABz-Gly-Arg-OMe-HCl (IV)—To a cooled proflavine. Dye binding is known to enhance the (0°C) solution of Arg-OMe-2HCl (3.13 g, 12 mmol) catalytic activity of papain toward small substrates in N, N-dimethylformamide (40 ml) and triethyl(10,11) and the rates at which the enzyme is in- amine (1.68 ml, 12 mmol) were added I (2.83 g, activated by N-alkylmaleimides (12). 10 mmol) and N-hydroxysuccinimide (1.15 g, 10 mmol), followed by N, N-dicyclohexylcarbodiimide (2.06 g, 10 mmol). The solution was stirred at EXPERIMENTAL 0°C for 2 h and then at room temperature over/•-Phenylazobenzol chloride (PABz-O) was pre- night. After filtration, the filtrate was poured into pared according to Anspon (13). The synthesis of 90% saturated NaCl solution (500 ml). The presubstrates is briefly described below. Optical cipitate wasfilteredoff, washed with 90% saturated rotations were measured on a Union Giken PM-71 NaCl solution, and dried over P,O6. The residue high sensitivity polarimeter. The solvent systems was dissolved in hot ethanol (EtOH) and, after used for thin-layer chromatography (tic) and paper removal of NaCl by filtration, the solution was chromatography (pc) were: solvent a, 1-butanol- evaporated. This treatment was repeated several acetic acid(AcOH)-water ( 4 : 1 : 5 , v/v, upper times to ensure complete removal of NaCl. The phase); solvent b, 1-butanol-AcOH-pyridine- residue was then scratched repeatedly with fresh dioxane to removed unreacted PABz-Gly. Finally water ( 4 : 1 : 1 : 2 , v/v). PABz-Gly (1)— To a solution of Gly (5.27 g, the residue was crystallized by treatment with ethyl 70.2 mmol) and NaHCO, (13.8 g, 164 mmo!) in ether. Recrystallization of the crude product was •water (50 ml) and dioxane (50 ml), a solution of repeated twice from EtOH-ethyl ether to afford PABz-Cl (11.5 g, 47 mmol) in dioxane (80 ml) was 2.70 g (55%) of crystals which melted at 228-230°C added dropwise for 15 min. Eventually crystals after softening at 102-104°C; [a] D -3.2° (c 2.0, deposited. After stirring for 5 h, the reaction mix- MeOH); Rf^ (a) 0.35, R,v* (a) 0.89. Anal. Calcd. ture was evaporated to remove dioxane, diluted for C H . ^ N j - H C l - H . O : C, 52.01; H, 5.95; N, with water (600 ml), and warmed to 60°C to obtain 19.30. Found: C, 52.42; H, 5.96; N, 19.10. /. Biochem.

STUDIES ON PAPAIN CATALYSIS

647

PABz-Glyt-Arg-OMe-HCl (V)— This was pre- were withdrawn, acidified to pH 2 with 1 M HC1 pared from II and Arg-OMe • 2HC1 in the manner and lyophilized. The dried residues were analyzed described for IV in a yield of 31 % and melted at by tic, pc, and paper electrophoresis. Paper 236-238°C after softening at 108-110°C; [ah — electrophoresis was carried out under the following 14.3° (c 2.0, MeOH), R,t* (a) 0.30, R,v (b) 0.92. conditions: buffer, AcOH-pyridine-water (10 : 20 : Anal. Calcd. for C ^ H ^ N , • HC1 • 2H.O: C, 49.44; 970, v/v) (pH 5.2); voltage gradient, 15V/cm; H, 6.05; N, 19.22. Found: C, 49.34; H, 5.90; period, 65 min; paper, Toyo No. 52. N, 18.98. Kinetics—Buffer solution was prepared with PABz-Gly-Arg (VI)—-To a solution of IV oxygen-free water. Concentrations of substrates (20.3 mg, 0.041 mmol) in MeOH (0.5 ml), 0 . 1 M were determined spectrophotometrically using the NaOH (0.5 ml) was added and the solution was left following molar extinction coefficients for PABzto stand at room temperature. The progress of Gly: Jsi4 (0.1 M phosphate buffer containing 3% saponification was checked by tic. The reaction DMSO, pH 6.5) 21,200 M-^crrr 1 ; tlt3 (MeOH) was complete in 2 h. The solvents were removed 25,000 M-1-cm"1. by lyophilization and the dried residue was stored The initial rates of hydrolysis by activated at -20 o C;.R/i