On the specificity of Bacillus subtilis neutral protease in relation to the compound active site

ARCHIVES

OF

BIOCHEMISTRY

AND

On the Specificity

BIOPHYSICS

of Bacillus

136, 311-323 (1969)

subtilis

Neutral

the Compound KAZUYUKI Shionogi

MORIHARA, Research

Laboratory,

Received

Active

TATSUSHI Shionogi

Protease

OKA,

to

Site AND

and Co., Ltd.,

June 3, 1969; accepted

in Relation

HIROSHIGE

Fukushima-ku,

September

TSUZUKI Osaka,

Japan

26, 1969

A previous paper indicated that a neutral protease of Bacillus subtilis has a large active site which can be divided into at least six subsites SrSa and S1’-Sa’, on both sides of the catalytic site. Each subsite accomodates one amino acid residue of a peptide substrate, and is numbered S,, Sz, etc. towards the NHz-end and S,‘, SZ’, etc. toward the COOH-end. To investigate the correlation between these six subsites and the specificity of the enzyme, kinetic studies were made using various synthetic peptides as substrates. The results led to the conclusion that the specificity is not always determined by all the subsites. The three subsites S,, S,‘, and SZ’ were the ones mainly concerned with the development of specificity, while the others were not always implicated.

The specificities of some proteases are known to be determined not only by the amino acid residues immediately surrounding the site of attack in the substrate but also by neighboring residues, as seen in pepsin (1, 2) and streptococcal protease (3), for example. Recently, Schechter et al. (4-6) estimated the size of the active site in both papain and carboxypeptidase A, and they proposed the hypothesis that the reactivity of a given peptide bond in a protein substrate depends on the nature of the several amino acid residues which interact with the active site in these enzymes. The specificity of pepsin and streptococcal protease may also be related to the size of the active site. However, no detailed study has been attempted to clarify the correlation between enzymatic specificity and the size of the active site in these enzymes. We have shown (7, 8) that a neutral protease from Bacillus sz$tilis has a large active site, exceeding 21 A in length, which can be divided into at least six subsites S,S, and S<-SQI, on both sides of the catalytic site. This conclusion was deduced from a study using various synthetic peptides as substrates and from the assumptions pre-

viously made by Schechter and Berger (4). The amino acid residues of a peptide substrate situated toward the NHz-end from the cleavage site, numbered as PI, Pz, etc., occupy adjacent subsites S1, Sz, etc., respectively; while those toward the COOH-end, numbered as PI’, P,‘, etc., occupy adjacent subsites &‘, S,‘, etc., respectively. The specificity of the enzyme is not only determined by subsite S,‘, which interacts with the amino acid residue containing the amino group to be attacked, as assumed in our previous papers (9, lo), but is determined by all the subsites which constitute the large active site, To clarify this concept, further experiments were undertaken using various new synthetic peptides as substrates.

311

EXPERIMENTAL

PROCEDURE

Materials A neutral protease (I X crystallized) from B. subtilis var. amylosacchariticus Fukumoto was Ltd. obtained from Seikagaku-Kogyo Co., (Tokyo). Various synthetic peptides such as ZGly-X-NH2 (X = L-serine, L-alanine, L-valine, n-isoleucine, L-leucine, n-phenylalanine, and Ltyrosine; Z = benzyloxycarbonyl), Z-A-Leu-NH2 (A = D- and L-alanine, L-proline, L-tyrosine, and

312

MORIHARA,

OKA,

n-phenylalanine), Z-Gly-Pro-Leu-Gly, Z-Gly-ProLeu-Gly-Pro, Leu-NHZ, and Leu-Gly were obtained from the Peptide Center at the Institute for Protein Research of Osaka University. Peptides such as Z-Gly-Gly-NH2, Z-Tyr-Gly-NHp, Z-Tyr-Ser-NHS, Gly-Ala-l>4HB0, Leu-Ala-HsO, Leu-Leu-acetate, Phe-Ala, Val-Ala, and AlaAla->sH20 were obtained from the Cycle Chemical Corporation, California. Phe-NH, was kindly supplied by Dr. M. Ebata of this laboratory. ZPhe-Leu-Ala and Phe-Leu-Ala-HCl were prepared according to the method described previously (8). The other peptides used were synthesized as described below. All the peptides were analyzed for carbon, hydrogen, and nitrogen and checked for purity by thin-layer chromatography (methanolchloroform-acetic acid, 10:90:3 or X1:80:3, v/v). Except when specified, the constituent amino acids were all of the n-configuration. Abbreviated designations of peptides or their derivatives obey the tentative rules of the IUPAC-IUB Commission on Biochemical Nomenclature.

Methods The extent. of hydrolysis of various synthetic peptides was determined as follows: A reaction mixture (1 ml) containing 0.05 M Tris buffer of concentrapH 7.0, 2.5 rnM CatI&, an appropriate tion of peptide and a suitable amount of enzyme was incubated at 40’. At various intervals, 0.1 ml of the reaction mixture was withdrawn and put into a test tube which contained 1 ml of a mixture of 0.5 M citrate buffer (pH 5) and 0.01 M EDTA solution to stop further hydrolysis. The extent of hydrolysis was measured by the ninhydrin met.hod of Yemm and Cocking (11). The ninhydrin color TABLE PERCENTAGE

COLOR

Compound

Color yield (%)

Gly-NH, Ser-NH2 Ala-NH* Val-NH2 He-NH2 Leu-NH, Phe-NH2 Tyr-NH2 Gly-Ala Ser-Ala Ala-Ala

65.1” 86.5” 75.8 10.5 12.05 59.0 65.0 78.3 91.8 84.gc 122.0

YIELDS V

0.28 0.31 0.38 0.54 0.64 0.64 0.66 0.60 0.24 0.31 0.31

AND

TSUZUKI

yields of the products, based on n-leucine as lOO~c, are shown in Table I. The ninhydrin values of Leu-n-Ala and Leu-Ala-n-Ala were assumed to be the same as those of the corresponding L-peptides. The ninhydrin color yields of Gly-NH2, Ser-NHz, Ser-Ala, Ile-Ala, Tyr-Ala, Leu-Gly-Pro, and LeuAla-Ala were deduced after complete hydrolysis of an equimolecular amount. of the precursor peptides by the B. subtilis enzyme or by another suitable enzyme; for example, the yield of GlyNH* was obtained from the hydrolysis of Z-TyrGly-NH2 by the enzyme or by subtilisin BPN’ (Nagarse). The sites of action of the enzyme upon substrates were determined by paper chromatography of the hydrolyzates with reference to authentic compounds, or by the usual DNPmethod. In all cases, satisfactory Michaelis-Menten kinetics were observed, and plots of l/S va. l/v (Lineweaver-Burk plot) permitted the fitting of definite straight lines. For each determination of Km and V,,,., derived from such plots, initial rates were measured from four (or more) values of the initial substrate concentration S. Due t,o the low solubility of most peptides presented here, determinations were made at lower concentrat.ions than the K, values of these substrates, in presence or absence of 2Oye ethanol. Depending upon the rate of cleavage, the enzyme concentration was adjusted suitably; it was assumed that 1 mg of the enzyme = 0.0296 pmole (mol wt, 33,800 (12)). This enzyme concentration was used to calculate k,,t from Vm., values.

Synthesis of Peptides Z-Ala-NH2. was prepared

A mixed carboxylic acid anhydride as usual (below -5”) from Z-Ala

I

OF COMPOUNDS

BASED Compound

Val-Ala Ile-Ala Leu-Ala Phe-Ala Leu-Gly Tyr-Ala Leu-Leu Leu-Gly-Pro Leu-Ala-Ala Phe-Leu-Ala Gly-Phe-Leu-Ala

ON L-LEUCINE

=

lCKF@

Color yield (%)

R?

12.5 12.8” 80.4 102.7 59.0 70.0” 100.0 65.0c 69.0” 101.8 20.8

0.53 0.68 0.67 0.63 0.51 0.55 0.88 0.49 0.63 0.85 0.76

0 The color yields were obtained on heating for 15 min at lOO”, and by using a 570 rnp filter. bn-Butanol:Acetic acid:Pyridine:Water (30:6:20:24, v/v). c The value was deduced from the complete hydrolysis of the corresponding peptide by the B. subtilis enzyme presented here, or by another suitable enzyme.

SPECIFICITY

OF B. subtilis

(5.37 mmole) and ethyl chloroform&e (5.44 mmole) in the presence of triethylamine (5.50 mmole), with tetrahydrofuran (35 ml) as the solvent. To the solution, corm. NHIOH (89.0 mmole) was added, and the reaction mixture was stirred for 20 min in the cold and then for 2.5 hr at room temperature. The product was crystallized from ethanol-ether-petroleum ether. Yield, 28%; mp 132-133”. Analysis. Calcd for CLHl*OaNs (222.2): C, 59.45; H, 6.35; N, 12.60. Found: C, 59.69; H, 6.28; N, 12.51. Ala-NHt-HCl. Catalytic hydrogenolysis (palladium black) of Z-Ala-NH2 (1.46 mmole) in the presence of cont. HCl (1.46 mmole) gave the product, which was crystallized from methanolethanol-ether. Yield, 97%; mp 218-220” (dec). Analysis. Calcd for C3H90N&l (124.6) : C, 28.93; H, 7.28; N, 22.49; Cl, 28.46. Found: C, 29.00; H, 7.43; N, 21.48; Cl, 27.79. Z-Val-NHS. A mixed carboxylic acid anhydride, prepared from Z-Val (6.11 mmole), was treated with NH,OH (89.0 mmole) as above. The product was crystallized from ethanol-ether-petroleum ether. Yield, 75%; mp 24X-207’. Analysis. Calcd for ClzHlsOsNz (250.3) : C, 62.38; H, 7.25; N, 11.19. Found: C, 62.43; H, 7.12; N, 11.32. Val-NH2-HCl. Catalytic hydrogenolysis of Z-Val-NH2 (4.92 mmoles) gave the product, which was then crystallized from ethanol-methanol-ether. Yield, 98$&; mp > 220”. Analysis. Calcd for CsHraON&l (152.6): C, 39.35; H, 8.59; N, 18.35; Cl, 23.23. Found: C, 39.57; H, 8.64; N, 18.47; Cl, 23.11. Z-Ile-NH2. A mixed carboxylic acid anhydride, prepared from Z-Ile (5.44 mmole), was treated with NHdOH (118 mmole) as above. The reaction mixture was dried in vacq and the residue was successively washed with 1% ammonia and water, and then dried. Yield, 80%; mp 224-225”. Analysis. Calcd for C~~HLWO~N~ (264.3): C, 63.62; H, 7.63; N, 10.60. Found: C, 63.63; H, 7.55; N, 10.87. Ile-NH2-HC1. Catalytic hydrogenolysis of ZIle-NH2 (4.37 mmole) gave the product, which was then crystallized from methanol-ether. Yield, 96.77;; mp >230° (sublimation). Analysis. Calcd for CeHisON&l (166.7): C, 43.24; H, 9.07; N, 16.90; Cl, 21.28. Found: C, 43.41; H, 9.02; N, 16.77; Cl, 21.55. N-Z-Tyr-NH2. A mixed carboxylic acid anhydride, prepared from N-Z-0-Ac-Tyr (28.8 mmoles), was treated with NHdOH (460 mmole) as above. The product was crystallized from methanol-ethylacetate-ether. Yield, 67.0yo; mp 145-146’. Analysis. Calcd for C1rHra0dN2 (314.3): C,

NEUTRAL

PROTEASE

313

64.96; H, 5.77; N, 8.91. Found: C, 65.07; H, 5.82; N, 9.05. Tyr-NH2-HCI. Catalytic hydrogenolysis of N-Z-Tyr-NH2 (17.8 mmole) gave the product, which was then crystallized from methanolethanol-ethyl acetate-ether. Yield, 99%; mp 226-228” (dec). Analysis. Calcd for C9Hi302N&l (216.7): C, 49.89; H, 6.05; N, 12.93; Cl, 16.36. Found: C, 49.68; H, 6.30; N, 12.87; Cl, 16.53. Z-Gly-AZa-OMe. Z-Gly-ONP (25.5 mmoles) and Ala-OMe, prepared from the hydrochloride (25.5 mmole), were coupled in the usual manner (2), with CHzCll (100 ml) as the solvent. Yield, 60$&; mp 91-92”. Analysis. Calcd for ClbHi805N2 (294.3): C, 57.14; H, 6.16; N, 9.52. Found: C, 57.04; H, 6.13; N, 9.53. Z-Gly-Ala. The above ester (5.42 mmole) was saponified as described in the previous paper (2). The product was crystallized from ethyl acetatepetroleum ether. Yield, 88%; mp 117-118”. Analysis. Calcd for CiaHl,OrN, (280.3): C, 55.71; H, 5.75; H, 9.99. Found: C, 55.81; H, 5.83; N, 9.83. Z-Phe-Gly-Ala. Z-Phe-ONP (4.54 mmole) and Gly-Ala, prepared by catalytic hydrogenolysis of Z-Gly-Ala (4.74 mmole), were coupled in the usual manner (2), with dimethylformamide (30 ml) and water (5 ml) as the solvent. The product was crystallized from ethanol-ethyl acetate-ether. Yield, 75%; mp 177-178”. Analysis. Calcd for C~~H~~OBN~ (427.5): C, 61.82; H, 5.90; N, 9.83. Found: C, 61.76; H, 5.78; N, 10.02. Z-Ser-Ala-OBz. Z-Ser-Na was prepared from the hydrazide (6.0 mmole) in the usual manner, with NaNOz (6.0 mmole) in 1.5 ml of distilled water in the presence of 6.0 ml CH&N and 9.0 ml of 2 N HCl below 5”. To the reaction mixture, a CH&N solution (6.0 ml) of Ala-OBz-TosH (6.0 mmole) was added in the cold (below 5”), and the pH was maintained at about 8 with triethylamine. The mixture was kept for one night in the cold. The reaction mixture was then concentrated to dryness, and the residue was taken up in ethyl acetate; the solution was successively washed with N HCl, 5% NaHCOa, and water, and dried over Na2SOk, and concent,rated in vacua. The product was crystallized from ethyl acetate-ether. Yield, 66%; mp 114.5-115.5”. Analysis. Calcd for CLH2406N2 (400.4): C, 62.99; H, 6.04; N, 7.00. Found: C, 63.11; H, 6.03; N, 7.22. Z-Phe-Ser-Ala. Z-Phe-ONP (3.30 mmole) and Ser-Ala, prepared by catalytic hydrogenolysis of Z-Ser-Ala-OBz (3.50 mmole), were coupled in the usual manner, wit.h dimethylformamide (20 ml),

314

MORIHARA,

OKA,

CH&ls (5 ml) and distilled water (3 ml) as the solvent. The product was crystallized from ethyl acetate-ether. Yield, 13%; mp 150-152”. Analysis. Calcd for C23H2707N3 (457.5): C, 60.39; H, 5.95; N, 9.19. Found: C, 59.82; H, 6.07; N, 8.77. Z-Ala-Ala-OMe. A mixed carboxylic acid anhydride was prepared from Z-Ala (40.0 mmole) and ethyl chloroformate (45.0 mmole )in the usual manner (below -5’), with CH&lz (100 ml) as the solvent. To the cooled solution, a CH&12 solution (120 ml) of Ala-OMe, prepared by neutralization of the hydrochloride (40.0 mmole) with cold triethylamine, was added. The product was crystallized from ethyl acetate-ether-petroleum ether. Yield, 64%; mp 104-105.5’. Analysis. Calcd for GsH~~O~N~ (308.3): C, 58.43; H, 6.54; N, 9.09. Found: C, 58.36; H, 6.61; N, 9.23. Z-Phe-Ala-Ala-OMe. A mixed carboxylic acid anhydride was prepared from Z-Phe (5.02 mmole) and ethyl chloroformate (5.26 mmole) in the usual manner, with CH&ls (30 ml) as the solvent. To the solution, a CH&L solution (25 ml) of AlaAla-OMe, prepared by catalytic hydrogenolysis of Z-Ala-Ala-OMe (5.02 mmole) was added. The reaction mixture was concentrated in vacua, and the residue was washed successively with N HCl, 5% NaHCOa, and distilled water, and was dried. Yield, 89%; mp 197-199”. Analysis. Calcd for CtdHpgOaNa (455.5): C, 63.28; H, 6.42; N, 9.22. Found: C, 63.54; H, 6.59; N, 9.18. Z-Phe-Ala-Ala. The above ester (3.99 mmole) was saponified as usual. After adjusting the pH at 6-7, the reaction mixture was concentrated in vacua. The residue was washed with N HCl and dissolved in 5% NaHCO.+ The acidification (about pH 1) gave the product, which was washed with distilled water and dried. Yield, 80%; mp 187.5188.5”. Analysis. Calcd for C23H2706N3 (441.5): C, 62.57; H, 6.16; N, 9.52. Found: C, 62.81; H, 6.37; N, 9.56. Z-Val-Ala-OMe. A mixed carboxylic acid anhydride was prepared from Z-Val (6.67 mmole) and ethyl chloroformate (7.37 mmole), with CHzClz (30 ml) as the solvent. To the solution, a CHzClz solution (30 ml) of Ala-OMe, prepared by neutralization of the hydrochloride (6.89 mmole) with cold triethylamine, w&s added. The product was crystallized from CH&lz-petroleum ether. Yield, 92.6%; mp 169.5-170.5”. Analysis. Calcd for G~H~~OSN~ (336.4): C, 60.70; H, 7.19; N, 8.33. Found: C, 60.72; H, 7.20; N, 8.58. Z-Phe-Val-Ala-OMe. A mixed carboxylic acid anhydride was prepared from Z-Phe (5.04 mmole)

AND

TSUZUKI

and ethyl chloroformate (5.26 mmole) as above, with CH&h (30 ml) as t.he solvent. To the reaction mixture, Val-Ala-OMe, prepared by catalytic hydrogenolysis of Z-Val-Ala-OMe (5.04 mmole), was added. The reaction mixture was dried in vacua, and the residue was washed successively with N-HCl, 5yo NaHC03, and water. Yield, 91%; mp 212-213”. Analysis. Calcd for CLBH~~O~N~ (483.6): C, 64.58; H, 6.88; N, 8.69. Found: C, 64.73; H, 6.92; N, 8.53. Z-Phe-Val-Ala. The above ester (4.06 mmole) was saponified as usual. Crystallization was achieved from CHCL-ether. The product was further purified by chromatography on a column of silica gel (CHCll-methanol system). Yield: 54%; mp 207-209”. Analysis. Calcd for C2~H3LOSN3 (469.5): C, 64.21; H, 6.68; N, 8.95. Found: C, 63.87; H, 6.60; N, 8.82. Z-Ile-Ala-OBz. Z-Ile (3.02 mmole) and Ala-OBz, prepared by neutralization of Ala-OBz-TosH (3.04 mmoles) with triethylamine, were coupled in the usual manner (2) in the presence of dicyclohexylurea (3.07 mmole), with CH&lz (30 ml) as the solvent. The product was crystallized from ether-CH&lz-petroleum ether. Yield, 89%; mp 165-166”. Analysis. Calcd for C2dH3,,05N2.j$H20 (435.5): C, 66.19; H, 7.17; N, 6.43. Found: C, 66.13; H, 7.11; N, 6.65. Z-Phe-Ile-Ala. Z-Phe-ONP (2.50 mmole) and Ile-Ala, prepared by catalytic hydrogenolysis of Z-Ile-Ala-OBz (2.70 mmole), were coupled in the usual manner, with dimethylformamide (20 ml) and distilled water (2 ml) as the solvent. The product was crystallized from ethyl acetateether-petroleum ether. Yield, 93%; mp 195-196”. Analysis. Calcd for C26H3306N3 (483.6): C, 64.58; H, 6.88; N, 8.69. Found: C, 64.32; H, 6.76; N, 8.78. Z-Phe-Ala-OMe. A mixed carboxylic acid anhydride was prepared from Z-Phe (6.67 mmole) and ethyl chloroformate (7.37 mmole) as above, with CH&lz (30 ml) as the solvent. To the solution, a CH&lz solution (20 ml) of Ala-OMe, prepared by the neutralization of the hydrochloride (6.75 mmole), was added. The product was crystallized from ethyl acetate-petroleum ether. Yield, 88.0%; mp 132-133”. Analysis. Calcd for C21HZd05N2 (384.4): C, 65.61; H, 6.29; N, 7.29. Found: C, 65.40; H, 6.24; N, 7.28. Z-Phe-Phe-Ala-OMe. A mixed carboxylic acid anhydride was prepared from Z-Phe (5.01 mmole) and ethyl chloroformate (5.26 mmole), with CH&l, (30 ml) as the solvent. To the solution, Phe-Ala-OMe, prepared by catalytic hydrogenoly-

SPECIFICITY

OF B. subtilis

sis of Z-Phe-Ala-OMe (5.00 mmole) was added. The reaction mixture w’~ts concentrated in vucuo, and the residue was washed successively with N HCl, 5% NaHC03, and distilled water, and was dried. Yield, 87%; mp 195-196’. Analysis. Calcd for CaoH,,O,N, (531.6): C, 67.78; H, 6.26; N, 7.90. Found: C, 68.05; H, 6.33; N, 7.79. Z-Phe-Phe-Ala. The above ester (3.62 mmole) was saponified as usual. The product was crystallized from methanol-ether. Yield, 77.0y0, mp 210211”. Analysis. Calcd for C29H3106N3.?$H20 (524.8) : C, 66.37; H, 6.11; N, 8.01. Found: C, 66.38; H, 6.09; N, 8.01. N-Z,O-Bz-Tyr-Ala-OBz. N-Z,O-Bz-Tyr-ONP (7.03 mmole) and Ala-OBa, prepared by neutralization of Ala-OBz-TosH (6.87 mmole) with triethylamine, were coupled in the usual manner. The product was crystallized from ethylacetatepetroleum ether. Yield, 62.7%; mp 140-142”. Calcd for C34H3406N2 (566.7): C, Analysis. 72.07; H, 6.05; N, 4.94. Found: C, 71.96; H, 6.01; N, 4.94. Z-Phe-T’yr-Ala. Z-Phe-ONP (2.85 mmole) and Tyr-Ala, prepared by catalytic hydrogenolysis of N-Z,O-Bz-Tyr-Ala-OBz (3.07 mmole), were coupled in the usual manner. The product was crystallized from methanol-ethyl acetate-ether. Yield, 50%; mp 197-198”. Analysis. Calcd for C29H310~N3 (533.6): C, 65.27; H, 5#.85; N, 7.87. Found: C, 64.90; H, 5.99; N, 7.69. Z-Phe-Leu-Gly-OEt. Z-Phe-ONP (7.82 mmole) and Leu-(fly-OEt, prepared by catalytic hydrogenolysis of Z-Leu-Gly-OEt (8.18 mmole), were coupled in the usual manner, with CH&lz (30 ml) as the solvent. The product was crystallized from ethyl acetate-methanol-ether. Yield, 87%; mp 154.5-155.0”. Analysis. Calcd for C&H3706N3 (497.6): C, 65.17; H, 7.09; N, 8.44. Found: C, 65.22; H, 6.78; N, 8.64. Z-Phe-Leu-Gly. The above ester (6.83 mmoles) was saponified as usual. The product was crystallized from ethyl acetate-ether-petroleum ether. Yield, 59%; mp 151-155”. Analysis. Calcd for Cz5H3106N3 (469.5): C, 63.95; H, 6.65; N, 8.95. Found: C, 63.80; H, 6.66; N, 8.86. Z-Phe-ONP (6.90 Z-Phe-Leu-D-Ala-OMe. mmole) and Leu-n-Ala-OMe, prepared by catalytic hydrogenol.ysis of Z-Leu-n-Ala-OMe (7.23 mmole), which w&s prepared as described previously (2), were coupled in the usual manner, with CH&lz (30 ml) ae the solvent. The product was crystallized from ethyl acetate-ether. Yield, 66%; mp 175-177”.

NEUTRAL

PROTEASE

315

Analysis. Calcd for C&H3hOBN3 (497.6): C, 65.17; H, 7.09; N, 8.44. Found: C, 65.11; H, 7.10; N, 8.36. Z-Phe-Leu-cAla. The above ester (4.56 mmole) was saponified as usual. The product was crystallized from ethyl acetate-ether. Yield, 93%; mp 175-176”; [~1nns.5-32.2 f 0.8” (0.8675y0 in methanol) Analysis. Calcd for C26H330~N3 (483.6): C, 64.58; H, 6.88; N, 8.69. Found: C, 64.61; H, 6.77; N, 8.56. Z-Phe-Leu-Leu-OEt. Z-Phe-ONP (7.58 mmole) and Leu-Leu-OEt,, prepared by catalytic hydrogenolysis of Z-Leu-Leu-OEt (8.23 mmole), which was prepared as described previously (2), were coupled in the usual manner, with CH.#& (30 ml) as the solvent. The product was crystallized from ethyl acetate-ether-petroleum ether. Yield, 73%; mp 103-105”. Analysis. Calcd for C&H4306N3 (553.7): C, 67.24; H, 7.83; N, 7.59. Found: C, 67.52; H, 7.81; N, 8.00. Z-Phe-Leu-Leu. The above ester (5.52 mmole) was saponified as usual. The product was crystallized from ethyl acetate-ether-petroleum ether. Yield, 97%; mp 8&81”. Analysis. Calcd for C29H,90aN3 (525.6): C, 66.27; H, 7.48; N, 7.99. Found: C, 65.97; H, 7.53; N, 8.26. Z-Gly-Phe-Leu-Ala. Z-Gly-ONP (3.57 mmole) and Phe-Leu-Ala, prepared by neutralization of the hydrochloride (3.62 mmole), were coupled in the usual manner, wit,h dimethylformamide (35 ml) and water (3 ml) as the solvent. The product was crystallized from methanol-ethyl acetateether. Yield, 69%; mp 194-196”. Analysis. Calcd for C2sHasO~N4 (540.6): C, 62.21; H, 6.72; N, 10.36. Found: C, 62.07; H, 6.55; N, 10.50. Gly-Phe-Leu-Ala-HC1. Catalytic hydrogenolysis of Z-Gly-Phe-Leu-Ala (0.37 mmole) gave the product, which was crystallized from methanolethanol-ethyl acetate-ether. Yield, 89%; mp > 220” (decomp.). Analysis. Calcd for C&H3105N&l (442.9): C, 54.23; H, 7.06; N, 12.65; Cl, 8.00. Found: C, 54.55; H, 7.21; N, 12.68; Cl, 7.65. Z-Ala-Phe-Leu-Ala. Z-Ala-ONP (3.52 mmole) and Phe-Leu-Ala (3.60 mmole), prepared as above, were coupled in the usual manner, with dimethylformamide (35 ml) and water (3 ml) as the solvent. The product was crystallized from methanol-ether. Yield, 58%; mp 224-226“; [~l]#~~ -55.2 f 0.8” (1.0168% in methanol). Analysis. Calcd for CJ~~H~~OTN~ (554.6): C, 62.80; H, 6.91; N, 10.10. Found: C, 62.90; H, 6.94; N, 10.02. Z-D-Ala-Phe-Leu-Ala. Z-D-Ala-ONP (3.48

316

MORIHARA,

OKA.

mmole) and Phe-Leu-Ala, prepared as above, were coupled in the usual manner, with dimethylformamide (10 ml), CII&l2 (25 ml), CHC13 (5 ml), CH,CN (2 ml), and water (2 ml) as the solvent. The product was crystallized from ethyl acetatect,her. Yield, B7yo; mp 194-196”; [LY]? - 12.3 f 0.5” (1.008~~ in methanol ilnalysis. Calcd for C~SIH&N~ (554.G): C, 62.80; H, G.91; N, 10.10. Found: C, 62.51; H, G.94; N, 10.24. Z-Phe-P&-&u-Ala, Z-Phe-ONP (3.80 mmole) and Phe-Leu-Ala, prepared as above, were coupled in the usual manner, with dimethylformamide (40 ml) and water (4 ml) as the solvent,. The product was crystallized from methanol-ether. Yield, G8%; mp 229-230”. Analysis. Calcd for C35H420TN4 (G30.7): C, 66.65; H, 6.71; N, 8.88. Found: C, 6G.17; H, 6.80; N, 8.72. Z-Leu-Ala-Ala-OMe. A mixed carboxylic acid anhydride was prepared from Z-Leu (13.0 mmole) and ethyl chloroformat,e (13.7 mmole), with CHzClz (50 ml) as bhe solvent. To the solution, a CH,Cl, solut,ion (40 ml) of Ala-Ala-OMe, prepared by catalytic hydrogenolysis of Z-Ala-Ala-OMe (13.0 mmole), was added. The product was cryst,allized from methanol-ether. Yield, 80%; mp 15G158”. Analysis. Calcd for CzlH,,06N, (421.5): C, 59.84; H, 7.41; N, 9.97. Found: C, 59.5G; I-I, 7.53; N, 10.19. Z-Phe-Leu-Ala-Ala-OMe. A mixed carboxylic acid anhydride was prepared from Z-Phe (2.40 mmole) and ethyl chloroformate (2.52 mmoles), wit,h CHeClz (15 ml) as the solvent. To the solution, a CH&lz solution (20 ml) of Leu-Ala-AlaOMe, prepared by catalyt,ic hydrogenolysis of Z-Leu-Ala-Ala-OMe (2.40 mmole), was added. The prodrlct, was crystallized from CH&la-ether. Yield, 890/c; mp 204-205”. Analysis. Calcd for C30H4007N4 (568.7): C, G3.3G; H, 7.09; N, 9.87. Found: C, 63.15; H, 7.19; N, 9.77. Z-Phe-Leu-Ala-Ala. The above ester (2.10 mmole) was saponified as usual. After neutralizat,ion with N HCl, the reaction mixture was concent,rated in D(CCWJ,and the residue was successively washed with N HCl, water, CHzC12, and CHC13. Yield, 75%; mp 213-215”; [o(W5 -38.4 f 0.7’ (l.O23$&in methanol). Analysis. Calcd for CS~HBO~N~.J~HQO (563.7) : C, 60.59; H, G.84; N, 9.75. Found: C, 60.33; H, 6.75; N, 9.95. 2.Ala-n-Ala-OMe. Z-Ala-ONP (30.0 mmole) and n-Ala-OMe, prepared from the hydrochloride (30.8 mmole), were coupled in the usual manner, with CH,C12 (100 ml) as the solvent. The product

ANI)

TSUZUKI

was cryst,allized from ethyl acetate-ether-petroleum ether. Yield, G6yo; mp 137-138”. Analysis. Calcd for C15H200~N2 (308.3): C, 58.43; H, 6.54; N, 9.09. Fourld: C, 58.37; H, 6.80; N, 9.33. Z-Leu-Ala-D-Ala-OMe. Z-Leu-ONP (9.77 mmole) and Ala-n-Ala-OMe, prepared by catalytic hydrogenolysis of Z-Ala-n-Ala-OMe (10.0 mmole), were coupled in the usual manner, with CH&X, (30 ml) as the solvent,. The product was crystallized from ethyl acetate-ether-petroleum ether. Yield, 750/,; mp 164.5-165.5”. .4nulysis. Calcd for C21H3106Nd (421.5): C, 59.84; H, 7.41; N, 9.97. Found: C, 59.57; H, 7.50; N, 10.21. Z-Leu-Atho-Ah. The above ester (7.37 mmole) was saponified as usual. The product was crystallized from methanol-ether. Yield, 83yo; mp 16% 170”. Analysis. Calcd for C2,,H2g06N3 (407.5): C, 58.95; H, 7.23; N, 10.31. Found: C, 59.00; H, 7.25; N, 10.55. %-Phe-LelL-.4la-D-Ala. Z-Phe-ONP (5.71 mmole) and Leu-Ala-D-Ala, prepared by catalytic hydrogenolysis of Z-Lerl-Ala-n-Ala (6.11 mmole), were coupled in the usual manner, with dimethylformamide (25 ml), CH,Cl:! (10 ml), and water (3 ml) as the solvent. The product was crystallized from methanol-ether. Yield, 647,; mp 204-205”; b1P j -46.3 + 0.9” (1.0077, in methanol). Analysis. Calcd for Cg8H380,N4 (554.6): C, 62.80; H, G.91; N, 10.10. Found: C, 62.26; H, 6.84: N, 10.27. RESULTS Kinetics with Z-Gly-X-NH,, Z-Tyr-X9HB, aml Z-Phe-X-Ala. A kinetic study was made using Z-Gly-X-NH,, Z-Tyr-X-NH2 and Z-Phe-X-Ala (X = various amino acid residues) as substrates. Fig. 1 shows the Lineweaver-Burk plot for each of these peptides, from which their kinetic parameters were derived as shown in Table II.’ It indicates that the K, values of Z-Gly-X-NH2 1 A previous paper (10) concerning the specificity of various neutral proteases from microorganisms noted that the rate of hydrolysis of Z-Gly-He-NH2 and Z-Gly-Val-NH2 was considT T erably smaller than that of Z-Gly-Leu-NH?. This T is inconsistent with the results presented here in Table II. This inconsistency is most likely due t,o t,he very loa ninhydrin values of both Ile-NH2 and Val-NH,. A re-examination (4 mM substrate,

SPECIFICITY

07

R. subtilis

(column I) were generally higher than those obtained by Feder (13), who determined the pararneters in a concentration of below 1 mM substrate using a pH-stat apparatus. Both the results with Z-Gly-X-NH, and Z-Phe-X-Ala (column III) indicate that the proteolytic coefficient (k,,,/K,) is markedly affected by the amino acid residue (X) in subsite S1’, as follows; L-leucine, L-isoleucinel :> L-phenylalanine, L-valinel > Lalanine >* L-serine, L-tyrosine, glycine. The table further indicates that Z-Tyr-XNH2 (column II) is much more sensitive than Z-Gly-X-NH2 when X is glycine or L-serine. Comparison of two peptides such as Z-Tyr-X-NH2 and Z-Phe-X-Ala (X = glycine or L-serine) indicates that the K, values of both are almost the same but that their k,,, differ by a factor of 70-140. This would mean that substitution of L-tyrosine or L-phenylalanine in place of glycine at subsite SI reduces the Km value, and substitution of L-alanine in place of amide causes art increase of l~,,~, independent of the nature of the amino acid residue occupying subsit,e S1’ (Table III shows that L-tyrosine and L-phenylalanine have almost the same affinity for subsite &). This view is further supported by the following: When Z-Gly-X-NH, and Z-Phe-X-Ala are compared, where X is the same residue, increased hydrolysis is observed, related to both the decrease of the Km value and the increase of k oat. This was also observed even when a n-amino acid residue occupied subsite Sl’. The hydrolysis of Z-Gly-n-Leu-NH, was too small to be measured but the hydrolysis of Z-Phe-n-Leu-Ala (the arrow shows the bond t split) was measurable and its proteolytic coefficient was 0.28 pM/minute/mg enzyme 4oj, methanol, 0.02 mg enzyme/ml reaction mixture, at pH 7 and 40”) showed that the percentage of hydrolysis at 20 min and at 10 hr by the B. subtilis enzyme was 23.6 and 95’%, respectively, in Z-Gly-Ile-NH2, 2 and 62y0, respectively, in Z-GlyVal-NH2, and 19 and 95%, respectively, in Z-GlyLeu-NH*. Other neutral proteases of bacterial origin also showed similar activities against both peptides. A. study of these enzymes will be reporkd in the near fut,ure.

NEUTRAL

PROTEASE

317

under the experimental conditions of 25 mM substrate and 5 % ethanol at pH 7 and 40”. Kinetics of Z-A-Leu-NH, and Z-PheLeu-B. The effect of differing amino acid residues at subsite S1or Sz’ was studied using Z-A-Leu-Ala and Z-Gly-Leu-B (A or B = t T various amino acid residues), as shown in the previous paper (8). The following experiment was undertaken to examine whether a similar phenomenon was observed when L-alanine at subsite Sz’ was replaced with an amide in the former peptide or when glycine at subsite S1 was replaced with L-phenylalanine in the latter one. Lineweaver-Burk plots for these peptides are shown in Fig. 2, from which the kinetic parameters were derived (Table III). Due to the remarkably low solubility of most of these peptides, determinations were made in presence of 20% ethanol, which decreases the koat value markedly (about l/10) but has little effect on Km in either Z-Gly-Leu-NH, or Z-Phe-Leu-Ala (refer to Table II). The table indicates that whereas in Z-ALeu-NH, (column I) the K, values are affected markedly, and the JCcatvalues but slightly, on altering A, the reverse is the case when B is altered in Z-Phe-Leu-B. The proteolytic coefficient (k,,,/K,,,) is affected by residues A or B as follows; L-tyrosine, L-phenylalanine > L-alanine (>) glycine >> L-proline, n-ala&e in subsite &, and L-alanine, L-leucine > glycine >> n-alanine in subsite Sz’. These results are completely analogous to those observed (8) with Z-ALeu-Ala and Z-Gly-Leu-B. Kinetics of Z-A-Phe-Leu-Ala and Z-PheLeu-Ala-B. The role of subsites Sz and Sa’ in the enzyme was studied using Z-A-GlyLeu-Ala

and Z-Gly-Leu-Gly-B

(A or B 2

various amino acid residues) as substrate, as shown in the previous paper (8). The present study was undertaken to investigate whether the role of subsites Sz and &’ was affected when the most effective amino acid residues occupied subsites S1, Sl’, and Sz’, respectively. For this purpose, Z-A-Phe-LeuAla and Z-Phe-Leu-Ala-B (A = glycine, D- or L-alanine, or L-phenylalanine; B = D- or L-alanine) were used as substrates.

MORIHARA. i

/

I

1

OKA, 1

- B IOO-

1

AND

TSUZUKI

'

. ./

I/CSl

(mid-‘)

FIG. 1. Lineweaver-Burk plots for the hydrolysis of Z-Gly-X-NH2, Z-Tyr-X-NH2 and Z-Phe-X-Ala. These peptides were all split at the peptide bond containing the amino group of X. The reaction mixtures contained 0.05 M Tris buffer (pH i’), 2.5 mM CaCh, a suitable amount of enzyme, and various concentrations of substrate (~2.5 mm) in presence of 1.7% ethanol. In both the cases of Z-Gly-Ala-NH2 and Z-Phe-Phe-NHz, the substrate concentrations used were -20 mu and ~0.77 mM, respectively. The enzyme concentration was adequately adjusted for each peptide, by which a linear relationship was observed at the initial velocity. The reaction was performed at 40”, and aliquots (0.1 ml) were removed at 3-min intervals for ninhydrin analysis. The ninhydrin values of Ile-NH2, Val-NHZ, Ile-Ala, and Val-Ala were remarkably small as seen in Table I; therefore 1 ml of the reaction mixture (5 ml) of each of the respective substrates such as Z-Gly-Ile-NH2, Z-Gly-Val-NHz, Z-PheIle-Ala, and Z-Phe-Val-Ala was used for the ninhydrin assay. Initial velocities were taken at 10 to 20% hydrolysis. The plots of Z-Gly-X-NH2 (A, B, C), Z-Tyr-X-NH2 (D), and Z-Phe-XAla (E, F, G) are as seen in the figure.

The determinations were carried out in presence of 20% ethanol because of the low solubility of most of these peptides. Of the peptides, Z-GIy;Phe;Leu-Ala, Z-Ala-Phe-Leu-Ala,

Z-Phe-Phe-Leu-Ala and T T Z-Phe>eu$a-Ala ( t shows the main g specificity and T shows the minor one) were cleaved at two peptide bonds under the conditions of the present study. On paper chromatography of the digests of these

peptides, (digested for 10 min under the conditions shown in Fig. 3), the spots produced by Phe-Leu-Ala or Ala-Ala were quite faint compared with those of Leu-Ala or Leu-Ala-Ala, respectively, (below l/10, by a densitometer). Thus, the kinetic parameters of these peptides were all determined by assuming that only one peptide bond, that involving the amino group of n-leucine, was split during hydrolysis. The Lineweaver-Burk plot for each of

SPECIFICITY

OF B. subtilis

NEUTRAL

TABLE KINETICS

OF Z-GLY-X-NH2,

319

PROTEASE

II

Z-TYR-X-NHz,

AND Z-PHE-X-ALA

Columns I, II, and III show the peptide groups of Z-Gly-X-NH2, Z-Tyr-X-NH2, and Z-Phe-X-Ala, respectively. The position of each amino acid residue in the peptide substrate is numbered PI, Pz, etc. to the NHZ- end and PI’, Pz’, etc. to the COOH- end from the point of cleavage which is shown by the arrow. The kinetic parameters of these peptides were derived from their Lineweaver-Burk plots in Fig. 1, which were determined in presence of 1.7% ethanol. Column

I

II

III

Peptide P2- P, f PI’-

ZZZZZZZZ-

GlyGlyGlyGlyGlyGlyGlyGly-

GlySerAlaValIleLeuPheTyr-

P2’

NH2 NH2 NH2 NH, NH, NH, NH2 NH,

Km (md Negligibly Negligibly

Negligibly

Gly- NH, Ser- NH,

50 50

ZZZZZZZZ-

GlySerAlaValIleLeuPheTyr-

50 50 6.6 12 3.7 3.7 0.86 15

Ala Ala Ala Ala Ala Ala Ala Ala

small reaction small reaction 0.4 1.1 20 20 0.14 small reaction

100 50 50 50 9.1

Z- TyrZ- TyrPhePhePhePhePhePhePhePhe-

Rest (se-‘)

these peptides is shown in Pig. 3, and the kinetic parameters derived from the figure are summarized in Table IV. The study of Z-Phe-Phe-Leu-Ala was difficult because of its remarkably small solubility. The hydrolysis was compared with that of Z-Phe-LeuAla under the experimental conditions of 1 mM substrate in the presence of 21.6% dimethylformamide at pH 7,40”. The results indicated that almost the same degree of hydrolysis occurred in both peptides. This and the above results indicate that the sensitivity of Z-Phe-Leu-Ala is only slightly increased even when the peptide chain is elongated towards the NH2- or COOH-end by glycine, L-alanine or L-phenylalanine. Some stereospecific selectivity was observed at both subsites Sz and S<, but the degree was much smaller than that found with Z-A-Gly-Leu-Ala or Z-Gly-Leu-Gly-B, as described previously (8). On the other hand, the hydrolysis of a Z-free peptide such as

0.004 0.022 0.4 0.4 0.015

0.2 0.4

0.004 0.008

14 56 80.5 loo0 1000 lOCKI 80.5 6

0.28 1.12 12.2 83.3 270.3 270.3 93.6 0.4

TABLE

III

OF Z-A-LEU-NH2 AND Z-PHE-LEU-B Columns I and II show the peptide groups of Z-A-Leu-NHr and Z-Phe-Leu-B, respectively. The kinetic parameters of these peptides were derived from Fig. 2, which was determined in presence of 20% ethanol.

KINETICS

COIUll”

I

II -

-

pz-

PI

Peptides f PI’- Pz’

Z- GlyZ- AlaZ- D-Ala-

Leu-NH2 Leu-NH* Leu-NH2

Z- ProZ- TyrZ- Phe-

Leu-NH2 Leu-NH2 Leu-NH2

ZZZZ-

Leu-Gly Leu-Ala Leu-D-Ala Leu-Leu

PhePhePhePhe-

100 2.5 0.05 50 I 5.0 i 0.1 Negligibly small reaction Small reaction 4 10 2.5 4 2.5 10 ~-~

-

4 8 2 3.8105.3 27.7 10 0.22 0.022 7.7 141.0 18.4

MORIHARA,

OKA,

AND

TSUZUKI

I / LSI I mM-‘I

2. Lineweaver-Burk plots for hydrolysis of Z-A-Leu-NH, and Z-Phe-Leu-B. These peptides were all split at the peptide bond containing the amino group of L-leucine. The reaction was carried out in presence of 20% ethanol because of the low solubility of most of these peptides, and the substrate concentrations used for Z-A-Leu-NH2 (A, B) and Z-PheLeu-B (C, D) are as seen in the figure. The other conditions for hydrolysis are described in Fig. 1. FIG.

I / CSI CmM-‘1 3. Lineweaver-Burk plots for the hydrolysis of Z-A-Phe-Leu-Ala and Z-Phe-LeuAla-B. The reaction was carried out in presence of 20y0 ethanol because of the low solubility of most of these peptides, and the substrate concentrations used for Z-A-Phe-Leu-Ala (A) and Z-Phe-Leu-Ala-B (B) are as seen in the figure. The other conditions are described in Fig. 1. FIG.

Gly-Phe-Leu-Ala was remarkably slight T compared with that of the corresponding Z-peptide (below l/50 by ninhydrin-assay at 5 MM substrate concentration). This was also observed in previous work (8). Kinetics of Z-Gly-Pro-Leu-Gly and Z-GlyPro-Leu-Gly-Pro. The hydrolysis of Z-ProLeu-NH2 was slow, as shown in Table III. A comparative study was made with the three peptides Z-Pro-Leu-NHz, Z-Gly-ProLeu-Gly, and Z-Gly-Pro-Leu-Gly-Pro, and

this is summarized in Table V. It was ascertained that cleavage of these peptides occurred at the Pro-Leu bond. As previously shown (8) glycine and amide at subsite Sz’ make almost the same contribution to hydrolysis. Thus, the difference in the activities of these peptides must be ascribed to the elongation of the peptide chain toward the NH,- or COOH-end. The kinetic parameters of the latter two peptides were determined (substrate concentrations, ~50 mM;

OF B. subtilis

SPECIFICITY

TABLE or Z-A-PHE-LED-AU

KINETICS

NEUTRAL

PROTEASE

321

IV AND

Z-PHE-LEU-ALA-B

Columns I and II show the peptide groups of Z-A-Phe-Leu-Ala and Z-Phe-Leu-Ala-B, respectively. The kinetic parameters of these peptides were derived from Fig. 3, which was determined in presence of 2Oyo ethanol. For comparison, the parameters of Z-Phe-Leu-Ala are presented. Column

I II

p3-

ZZZ-

Peptides P,’ -

Pr’ -

Phe-

Leu-

Ala

GlyAlaD-Ala-

PhePhePhe-

LeuLeuLeu-

Ala Ala Ala

ZZ-

PhePhe-

LeuLeu-

AlaAla-

P2-

PI

Z-

t

P3’

Ala D-Ala

Km b-4

kmt (se’)

hnt/Km

3.8

105.3

27.7

3.4 3.8 7.1

83.3 142.8 80.0

24.5 37.6 11.3

3.8 10.0

82.0 74.1

21.6 7.4

TABLE \ at pH 7 and 40”). The K, and kCat (se+) of Z-Gly-Pro-Leu-GIy were 60 rnM and 6.8, EFFECT OF A NEIGHBORING RESIDUE ON THE Hydrolysis OF A PEPTIDE BOND PRO-LEU respectively, and those of Z-Gly-Pro-LeuThe reaction mixture containing 0.05 M Tris Gly-Pro were 55 mM and 17, respectively. Under the same conditions, the K,,, and lCoat buffer (pH 7), 1 rn~ substrate, 2.5 mM CaClp, 10% dimethylformamide, and 0.1 mg enzyme per (se+) of Z-Gly-Leu-NH2 were determined ml was kept at room temperature. After 7 and 20 to be 50 mM and 51.2, respectively. DISCUSSION

The previous paper (8) indicated that the extent of hydrolysis of a peptide bond containing the amino group of L-leucine (corresponding to subsite S,‘) was doubled when effective amino acid residues occupied both subsites S, and Sz’. The ratios of the proteolytic coefficients in a single replacement were as follows: Z-Gly-Leu-Gly/Z-Gly-LeuAla (1: 38), Z-Gly-LeurNHz/Z-Ala-LeuTNH, (1: 3) and Z-G1yqTLeu-NH,!ZPheTLeu-NH, (1:30). In the double replacement,Tthe ratios of the proteolytic coefficient for Z-Gly-LeuGly to Z.-Ala;Leu-Ala

and Z-Phe;Le:-Ala

were found to be 1: 111 and 1:1241, respectively. This suggested that the ratio of the sensitivity of Z-A-Leu-B to that of Z-GlyT T Leu-Gly could be calculated by multiplying the two factors for A and B, which occupied subsites S1 and S2’, respectively. The factor for each amino acid residue A and B can be determined from the ratio of its proteolytic eo&icient to that of glycine at the respective subsite.

hr of incubation, determined. Pa- P2 -

P,

the percentage

Peptide f P,‘-

Pz’-

Pa’

ZPro- Leu- NH2 Z- Gly- Pro- Leu- Gly Z- Gly- Pro- Leu- Gly- Pro

of hydrolysis

was

I hr 20 hr (% of hydrolysis)

8.0 30.7 62.4

26.5 62.2 100.

The present data with either Z-A-Leu-NH2 T or Z-Phe-Leu-B (Table III) were compared with those for Z-A-Leu-Ala or Z-Gly-Leu-B, T T respectively, described in the previous paper (8), and suggest that A and B are not dependant upon the kind of residue that occupies subsite Sz’ or S1, respectively, when the sensitive peptide bond involves the amino group of L-leucine. The factor (A) in subsite S1 mainly relates to binding (Km), and (B) in subsite Sz’ relates to catalysis Qc,,~), as shown in this and the previous paper (8). A comparative study with Z-GlyX-NH,

or Z-Tyr-X-NH*

and Z-Phe-X-Ala

(Table II) indic&es that the reducion of the K,,, value by replacement of glycine by L-tyrosine or L-phenylalanine at subsite S1

322

-MORIHARA,

OKA. AND TSUZUKI

and the promotion of catalysis (keat) by the presence of n-alanine in place of amide at subsite S 2’ are of almost the same degree regardless of the nature of X. This possibly indicates that the factor X for subsite S1’, related to both binding (K,) and catalysis (IC&, is not dependant upon the kind of amino acid residue that occupies subsites S1 and Sz’. This also means that factors A or B in subsite S1 or S,‘, respectively, are not dependent on the kinds of amino acid residues at subsite Sl’. The deductions lead to the concept that the ratio of the sensitivity of Z-A-X-B to that of Z-Gly;Gly-Gly

can be calcuated

by

multiplying the three factors of A, X, and B. From the kinetic results with Z-A-Leu-Ala reported in the previous paper (8), or from those with Z-A-Leu-NH2 (italicized) in Table III, the following factors for amino acid residues at subsite S1 were determined: n-phenylalanine (33, 50)) n-tyrosine (50)) L-alanine (3, .2), glycine (1)) L-proline (<
The present study indicates that the kinetic parameters of Z-A-Phe-Leu-Ala and Z-Phe-Leu-Ala-B

are similar Tto those of

Z-PheILeu-Ala, when glycine, n-alanine or T n-phenylalanine are used as A or B. On the other hand, the prot,eolytic coefficients of Z-A-Gly-Leu-Ala and Z-Gly-Leu-Gly-B are T ? remarkably high compared with those of Z-Gly-Leu-Ala and Z-Gly-Leu-Gly, respectively: when an effective amino acid residue is used as A or B, but the activities do not exceed that of Z-Phe-Leu-Ala, as described in the previous paper (8). This may indicate that both binding and catalysis are almost complete (i.e., reach “saturation”) when all three subsites S1, Si’ and Sz’ are occupied by effective amino acid residues. This is the case with the peptide sequence -Phe-LeuAla-, and here the other subsites Sz, Sa and S3’ play an insignificant role. On the other hand, when a peptide sequence of -Gly-LeuGly- (or -Gly-Leu-Ala-) occupies subsites S1, S,’ and Si, both binding and catalysis are not complete, thus making the roles of the other subsites significant in the development of full activity. Z-Pro-Leu-NH2 is much less rapidly hydrolyled

by the enzyme than is Z-Gly-

Leu-NH, (Table III), indicating tha\ L-proline at subsite S1inhibits the hydrolysis. Comparison of Z-Pro-Leu-NH, with Z-GlyPro-Leu-Gly

or Z-Gly-Pro-Leu-Gly-Pro

(Table V) indicates that eloigation of the peptide chain to subsite Sz by glycine partly reverses the inhibition by L-proline at subsite &. When glycine is present at subsite S1, elongation of the peptide chain to subsite Sz by glycine rather decreases the sensitivity, as seen in the correlation between Z-GlyLeu-Ala

and Z-Gly-Gly-Leu-Ala described f in the previous paper (8). This may indicate that inhibition by an inadequate amino acid residue such as n-proline, n-amino acid, etc. in subsites S1, Sp or Sz’ is partly reversed by elongation of the peptide chain toward the NH2- or COOH-end of the substrate, in

SPECIFICITY

OF B. subtilis NEUTRAL

which case the other subsite participates in the hydrolysis. These concepts lead to the conclusion that the specificity of the enzyme is not always determined by all the subsites which constitute the large active site. TWO subsites such as S1and S1’ are essential for hydrolysis, as demonstrated by the fact that a dipeptide backbone is required for hydrolysis by the enzyme (10, 13). From inhibition studies (8) with Z-dipeptides, it is clear that both these subsites are concerned with the binding of substrate. The contribution of subsite Sz’ is also significant for the appearance of specificity, as discussed above, and mainly relates to catalysis. The other subsites Sz, Sa and Sg’ are not always involved with the determination of specificity, their contributions to hydrolysis being dependent upon the kind of amino acid residues occupying subsites S1, S,’ and Sz’, but these nonessential subsites do appear to exert some regulation of hydrolysis. ACKNOWLEDGMENT We are greatly indebted to Drs. H. Otsuka, K. Inouje and M. Shin of this laboratory, for their technical advice for the preparabion of various new synthetic peptides.

PROTEASE

323

REFERENCES 1. TANG, J., Nature 199, 1094 (1963). 2. INOUYE, K., AND FRUTON, J. S., Biochemistry 6, 1765 (1967). 3. GERWIN, B. I., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 241,333l (1966). 4. SCHECHTER, I., AND BERGER, A., Biochem. Biophys. Res. Commun. 27, 157 (1967). 5. ABRAMOWITZ, N., SCHECHTER, I., AND BERGER, A., Biochem. Biophys. Res. Commun. 29, 862 (1967). 6. SCHECHTER, I., AND BERGER, A., Biochem. Biophys. Res. Commun. 32,898 (1968). 7. MORIHARA, K., AND OKA, T., Biochem. Biophys. Res. Commun. 30,625 (1968). 8. MORIHARA, K., OKA, T., AND TSUZUKI, H., Arch. Biochem. Biophys. 132.489 (1969). 9. MORIHARA, K., Biochem. Biophys. Res. Commm. 26, 656 (1967). 10. MORIHARA, K., TSUZUKI, H., AND OKA, T., Arch. Biochem. Biophys. 123,572 (1968). 11. YEMM, E. W., AND COCKING, E. C., Analyst 80, 209 (1955). 12. TSURU, D., KIRA, H., YAMAMOTO, T., AND FUKUMOTO, J., Agr. Biol. Chem. (Tokyo) 80, 1164 (1966). 13. FEDER, J., Biochemistry 6,2088 (1967). 14. FEDER, J., AND LEWIS, C., JR., Biochem. Biophys. Res. Commun. 28, 318 (1967). 15. BENSON, A. M., AND YASUNOBU, K. T., Arch. Biochem. BioDhus. 126.653 (1968).