[50] Dipeptidyl carboxypeptidase from Escherichia coli

[50]

DIPEPTIDYL CARBOXYPEPTIDASE

599

D i s t r i b u t i o n of S i m i l a r E n z y m e s

DFP-inhibited carboxypeptidase have been isolated from a variety of sources: citrus peel, '-'~ citrus leaves, '-'3 yeast, '-'~ Aspuergillus saitoi, 1"~ Aspergillus oryzae, ",-~French bean leaves (phaseolain),26 germinating barley, -°~ germinating cotton seedlings, ~s tomatoes, 2'~ watermelons, 3° and bromelain powder21 Furthermore, DoP ~ has suggested that cathepsin A from pig kidney is identical with catheptic carboxypeptidase and possibly related to the plant and fungal carboxypeptidases. ~ H. Zuber, Nature (London,) 201, 613 (1964). :~B. Sprossler, H.-D. Heilmann, E. Grampp, and H. Uhlig, Hoppe-Seyler's Z. Physiol. Chem. 352, 1524 (1971). :~T. Hata, R. Hayashi, and E. Doi, Agric. Biol. Chem. 31,357 (1967). ~T. Nakadai, S. Nasumo, and N. Iguchi, Agric. Biol. Chem. 36, 1343, 1473, 1481 (1972). :~'W. F. Carey and J. R. E. Wells, J. Biol. Chem. 247, 5573 (1972). '-'~K. Visuri, J. Mikola, and T.-M. Enari, Eur. J. Biochem. 7, 193 (1969). :~J. N. Ihle and L. S. Dure, J. Biol. Chem. 247, 5034, 5041 (1972). :'~T. Matoba and E. Doi, Ag~ic. Biol. Chem. 38, 1901 (1974). ~°T. Matoba and E. Doi, Agric. Biol. Chem. 38, 1891 (1974). :~'E. Doi, C. Ohtsuru, and T. Matoba, J. Biochem. (Tokyo) 75~ 1063 (1974). '~:E. Doi, J. Biochem. 75, 881 (1974).

[50] D i p e p t i d y l

Carboxypeptidase

from Escherichia

coil

B g A. YARO~

Assay Methods Principle. Dipeptidyl earboxypeptidase (DCP) from E. coli is an intracellular enzyme, which hydrolytically removes dipeptides from the carboxyl end of low- and high-molecular-weight peptides. 1 Two methods for measuring the enzymic activity are described. Method 1: The assay used for monitoring the enzyme purification is based on the release of glycylproline from the carboxyl end of the sequential polypeptide D n p ( P r o - G l y - P r o ) , , using the colorimetric ninhydrin method for quantitative determination of the dipeptide.

I)np(Pro-Gly-Pro),~_l-Pro-Gly-Pro ~ l)np(Pro-Gly-Pro)n_,-Pro -~- Gly-Pro

(1)

where n is the average number of the repeating units P r o - G l y - P r o per molecule. 1A. Yaron, D. Mlynar, and A. Berger, Biochem. Biophys. Res. Commun. 47, 897 (1972).

600

EXOPEPTIDASES

[501

Only one molecule of Gly-Pro is formed from each polymer chain, since the release of the next dipeptide would require the cleavage of a secondary amide, to which a proline residue donates the nitrogen. Such bonds are resistant to hydrolysis by DCP. The sequential polymer is rich in proline and has no free amino groups. It is therefore resistant to most peptidases. The only known enzyme capable of hydrolyzing it is collagenase. ~ No interference by this enzyme was experienced with DCP preparations purified beyond the ammonium sulfate precipitation step of the purification procedure described below. Aminopeptidase p3 and a dipeptidase activity present in the extract of E. coli, both are capable to hydrolyze glycylproline to glycine and proline if incubated under the conditions used for the assay of DCP in the presence of Mn 2÷ ions. As the assay for DCP does not require the adding of cations, the presence of these enzymes does not prevent the quantitative determination of the activity of DCP by analysis of the Gly-Pro formed. However, if crude enzyme preparations are used, they should be dialyzed and/or EDTA (0.1 mM) should be added before the assay. In the purification procedure described below, aminopeptidase P is separated from DCP in the acetone fractionation step, and the dipeptidase activity by hydroxyapatite chromatography. Method 2: In the absence of interfering peptidases benzyloxycarbonyltetraalanine can be used as the substrate and the hydrolysis by DCP (Eq. 2) may be followed either by the colorimetric ninhydrin method (not described here) or by the potentiometric method, using a recording pH star. Z--Ala4--~Z--Ala2 + Ala2 (2) At pH 8.1, which is the pK of the amino group of alanylalanine,4 half a mole of base is consumed per mole of Z-Ala~ hydrolyzed.

The Colorimetric Ninhydrin Method Using DinitrophenylPoly (Prolyl-Glycyl-Prolyl) as the Substrate Preparation o] Dnp(Pro-Gly-Pro)n. This compound is prepared by dinitrophenylation of poly(Pro-Gly-Pro) ~ as follows: Fluorodinitrobenzene (300 rag) is dissolved in ethanol (20 ml) and the solution is added to an aqueous solution (10 ml) containing poly(Pro-Gly-Pro) (500 mg, 2E. Harper, A. Berger, and E. Katchalski, Biopolymers 11, 1607 (1972). A. Yaron and A. Berger, this series Vol. 19, p. 521. 4E. Ellenbogen, ]. Am. Chem. Soc. 74, 5198 (1952). 5j. Engel, J. Kurtz, E. Katchalski, and A. Berger, d. Mol. Biol. 17, 255 (1966). The polymer is commercially dvailable from Miles-Yeda Ltd., Kiriat Weizmann, Rehovot, Israel.

[501

DIPEPTIDYL CARBOXYPEPTIDASE

601

average molecular weight 1300) and sodium bicarbonate (252 mg). The solution, protected against light, is left at room temperature overnight, extracted with three 50-ml portions of ether and concentrated to a volume of 2.5 ml by ultrafiltration (UM-05 membrane, Diaflo ultrafiltration cell, Amicon Corp.). This solution is applied to a column (2.2 X 100 cm) of Sephadex G-15 and eluted with water at a flow rate of 30 ml/hr. The effluent is monitored at 360 nm and collected in 7-ml fractions. Most of the material appears as a major peak beginning immediately after the void volume and emerging in about 80 ml. Smaller peaks that follow are discarded, and fractions containing material from the major peak are pooled, lyophilized, and dried in vacuo over sulfuric acid. The yield is about 300 rag. The number average chain length (n) of the polymer used by us was 4.9. This was established by quantitative spectrophotometric determination of the Dnp end groups (~36onm = 16,800, phosphate buffer pH 7.0). Complete substitution of N-terminal amines by Drip was demonstrated by showing that the polymer is resistant to hydrolysis by clostridial aminopeptidase. 6 This enzyme releases one proline residue from the amino end of every unsubstituted chain of (Pro-Gly-Pro)~. Reagents

Substrate stock solution is prepared by dissolving Dnp(Pro-GlyPro)~ in water (7.2 mM, concentration is determined spectrophotometrically, c3Gonm = 16,800 M -1 cm-1) Veronal buffer, 0.05 M, pH 8.15 Sodium acetate buffer, 4 M, pH 5.3, containing 0.2 mM sodium cyanide, freshly prepared before the assay from 4.08 M sodium acetate and 10 mM sodium cyanide Ninhydrin, 3% in methyl Cellosolve (ethyleneglycol monomethyl ether) Isopropanol in water, 50% Enzyme, the enzyme solution is suitably diluted with the Veronal buffer to obtain a solution containing 0.2-1.0 unit/ml Procedure. The substrate stock solution (50 ~I) is mixed with Veronal buffer (0.95 ml) and placed in a water bath at 40°; the enzyme solution (10 t~l) is added. After 15 rain the incubation mixture is analyzed for ninhydrin colorJ To each tube, containing 1 ml of incubation mixture, the ninhydrin solution (0.5 ml) and the acetate-cyanide buffer (0.5 ml) are added. The tubes are heated in a boiling water bath for

E. Kessler and A. Yaron, Biochem. Biophys. Res. Commun. 32, 658 (1968); see also Eur. Y. Biochem. (1976), in press, and this volume [45]. 7H. Rosen, Arch. Biochem. Biophys. 60, 10 (1957).

602

EXOPEPTIDASES

[50]

20 min and cooled with tap water. A mixture of isopropanol and water (1:1 v / v ) is added and the mixture is well mixed. The optical density at 570 nm is determined and the amount of glycylproline is calculated from a calibration curve constructed with known amounts of the dipeptide. The absorption of Drip at 570 nm is small and is corrected by subtraeting the substrate blank. Definition of Unit and Specific Activity. One enzyme unit is defined as the amount of enzyme which produces 1 umole of glyeylproline per minute under the conditions of the above described assay. This is 10.3 larger than the previously defined unit. 1 The specific activity is expressed in units per milligram of protein. The rate of hydrolysis is proportional to enzyme concentration in the range from zero to about 17 mU/ml, but deviates from linearity at higher concentrations.

The Potentiometric Assay Reagents Substrate stock solution: a 10 m M solution of Z-Ala,~ in 0.1 M KC1 is prepared. A sufficient amount of 1 M K O H in 0.1 M KC1 is added to bring the pH to 8.1 Base, 2 rn3I K 0 H

Procedure. Assays are performed in a pH star. We used the Radiometer, Copenhagen assembly consisting of a pH meter Model 26 with a glass electrode, connected through an agar bridge to a calomel electrode. The titrant is delivered from a 0.25-ml burette of an automatic pipette ABU-12 controlled with a Titrator 11 and coupled to a recorder (Scrvograph R E C 51). The reaction vessel, thermostatted at 40 ° is equipped with a magnetic stirrer, and the titrated solutions are kept under a constant stream of CO2-free argon. The substrate stock solution (0.5 ml) and 0.1 M KC1 (0.5 ml) are placed into the reaction vessel and the pH is adjusted to 8.1. When therreal equilibrium is reached, there is no uptake of base. The enzyme solution is mixed in, and the uptake of base is recorded as a function of time. Initial rates are calculated from records with a time scale usually between 10 see/era and 60 sec/cm. Enzyme concentration is chosen so that the line recorded has an inclination of 15°-60 °. To obtain the number of mieromoles of substrate hydrolyzed per minute, the number of micromoles of base titrant consumed per minute is multiplied by 2, since 0.5 mole of base is consumed per mole of sub-

[50]

DIPEPTIDYL CARBOXYPEPTIDASE

603

strate hydrolyzed. One unit of D C P as defined by the ninhydrin colorimetric method was found to correspond to a hydrolysis rate of Z-Ala4 of 3.24 ~moles/min. The potentiometric assay can be used only in the absence of other peptidases. Purification Procedure The enzyme is isolated from E. coli B wild type (American type culture collection catalog No. 23226). The following procedure (see also Table I) results in a 1200-fold purification. Purification starts from 10 kg of packed ceils. The amount of pure enzyme obtained in a single run is limited by the capacity of the preparative polyacrylamide gel electrophoresis, which is the last purification step. All operations are carried out at 4 ° . Preparation of Cell-Free Extract. Mass cultures of Escherichia coli, strain B are grown for 5 hr in a 500-liter fermentor (Biotec, Sweden) with aeration, on Davis media, tryptone (0.6%) and glucose (0.2%). The cells are harvested in a Sharples centrifuge yielding 4 g of wet cells per liter. The wet cells are stored frozen at --20% The frozen mass (10 kg) is dispersed at 4 ° in a solution of 0.9% KC1 (38 liters) by occasional shaking during 2 days. DNase (2.5 rag) is added and the cells are ruptured by passing the cold suspension through a Manton-Gaulin homogenizer (Model 15 M, Everett, Massachusetts) at 5000 psi at a rate of 46 liters/ hr. Cell debris is removed from the extract by centrifugation, using a Westphalia Separator AG, Model K D D 605 at 10,000 rpm and a flow rate of 12 liters/hr. After each 10 liters of suspension that pass, the sediment filling the head of the centrifuge is removed. The total weight of packed sediment is 7 kg. The effluent emerges at 20o-23 ° when running tap water is used to cool the centrifuge. The colleeted solution (40 liters) is stored at 4 ° for 15 hr. Heat Treatment. The cell-free extract is rapidly warmed to 50 ° by passing it through a spiral glass tubing of 0.5 cm internal diameter and 125-ml capacity that is immersed in a water bath (50 liters) kept at 51 ° with two B. Brown, Thermomix II immersion thermostats. A finger pump (Sigma Motor Model T8) is used to keep a flow rate of 200 ml/hr. The solution emerges at 50 ° and is kept at that temperature for about 15 rain by continuing the flow through a 30-meter Tygon tubing of 1.12 cm internal diameter immersed in a 50 ° water bath. The temperature of the suspension is now lowered by passing it through additional 7 meters of Tygon tubing immersed in an ice bath. The effluent emerging at 28 ° is collected and stored at 4 ° for about 9 hr. For determination of enzymic activity, the supernatant is first dialyzed against 0.9% KC1.

604

EXOPEPTIDASES

[~0]

~.~-~

~

~

~

~ ~ o

"~

.<

•~

~NZ

©

~

~.~

0

g

>

~

~

~.~

© Z © ~,.~

2 r~

e

~~ ~ ,

g.g

~

o

o " ~ ~" ~

•~ : ~.~'~

~ ~ ~",~

~-~

~

.~

[50]

DIPEPTIDYL CARBOXYPEPTIDASE

605

The same results are obtained for activity with nondialyzed solutions, but blanks are much higher. Ammonium Sul]ate Fractionation. The suspension from the previous step is divided into two equal parts, and each is treated separately. Saturated ammonium sulfate solution s (13.3 liters) is added at 4 ° to the suspension (20 liters) under mechanical stirring. After 90 min at 4 °, the solid material (1.5 kg of packed precipitate) is removed by centrifugation, using the Westphalia separator at 10,000 rpm, at a flow rate of 15 liters/hr. To each liter of the stirred supernatant, saturated ammonium sulfate solution (0.5 liter) is added, stirring is continued for 2 hr at 4 °, and the precipitate formed is collected by centrifugation (Sharpies Super Centrifuge 1A, open-type turbine, 40,000 rpm, 15 liters/hr). The precipitate obtained from the two runs is dissolved in 0.05 M sodium acetate, pH 5.6 (7 liters), and subjected to acetone precipitation without delay. For determination of protein and activity of the enzyme a sample is dialyzed against 0.05 M sodium acetate, pH 5.6. Acetone Fractionation. Cold acetone (kept at --20 ° prior to use, 5.6 liters) is added slowly (over 1 hr) to the solution obtained in the previous step (7 liters). The mixture is kept at 8°-11 ° by cooling with an external ice-salt bath. Stirring is continued at 8°-11 ° for 20 min, and the precipitate (about 250 g) is removed with a continuous centrifuge (Westphalia Separator, 10,000 rpm, 20 liters/hr). Cold acetone (4.2 liters) is added to the supernatant (12.5 liters) during about 20 rain, keeping the temperature of the well mixed solution at 8°-11 ° . Stirring is continued for an additional hour, and the precipitate formed is collected by centrifugation (Sharpies Super Centrifuge 1A, open-type turbine, 40,000 rpm, 30 liters/hr). About 70-80 g of packed precipitate is obtained. This is dissolved in 0.05 M sodium acetate, pH 5.6 (1 liter), the yellow solution obtained is dialyzed against 0.005 M phosphate buffer, pH 6.0 (final volume is 1130 ml), and stored at --20 °. At this stage the enzyme is quite stable, about 9% of activity being lost in 1 month. Ion-Exchange Chromatography on DEAE-Cellulose. The dialyzed solution from the previous step (1100 ml) is applied under gravity (90 ml/hr) to a 11.5 X 28.5 cm bed of DEAE-eellulose (Whatman DE 23) equilibrated with 0.01 M sodium phosphate, pH 6.0. The column is then washed with 4 liters of 0.01 M sodium phosphate, pH 6.0, 0.05 M in KC1, and eluted with a 36-liter linear gradient to 0.24 M KC1 in 0.01 M sodium phosphate, pH 6.0. Fractions of 200 ml are collected at 1200 ml/hr, and the effluent is monitored at 280 ran. Fractions between 15,090 and s The saturated ammonium sulfate solution was prepared as described previously by Yaron and Berger."~

606

EXOPEPTIDASES

[50]

18,660 ml, containing the activity, are combined, concentrated to 20 ml by ultrafiltration (Amicon PM-30 membrane), and stored frozen at --20% No loss of activity was observed after 20 days of storage. Gel Filtration on Sephadex G-150. The above concentrated solution (19 ml) is applied to a 2.8 }( 245 cm column of Sephadex G-159 (Pharmacia) preequilibrated with 0.1 M sodium phosphate, pH 6.0. The same buffer is used for elution, 6.7-ml fractions being collected at 13.5 ml/hr. The active fractions are combined (113 ml) and stored at --20% A 20% loss of activity was observed after 5 months of storage. Hydroxyapatite Column Chromatography. An aliquot of the above solution (24 ml) is applied under gravity to a hydroxyapatite' column (1.3 X 92 cm) equilibrated with 0.01 M sodium phosphate, pH 6.0. A 2-liter linear gradient from 0.01 M to 0.08 M sodium phosphate, pH 6.0, is used for elution. Fractions of 11 ml are collected at a flow rate of 24 ml/hr. The active fractions are concentrated to 12 ml by nltrafiltration (PM-30 membranes). The solution is kept frozen at --20% Polyacrylamide Gel Electrophoresis. Samples from the previous purification step (150 t~l) containing about 100 ~g of protein and 20% sucrose, are applied to 7% gels (6.5 }( 50 ram). The gels are prepared and the electrophoresis is performed according to Davis, ~° omitting the spacer gel, and using the Shandon disc electrophoresis appartus. Prior to application of the sample a current of 3 mA per gel is applied for 25 min. Separation is achieved by applying 3 mA per gel for 90 min at 4 °, six gels being used at a time. The location of proteins in the gel is detected spectrophotometrically at 280 nm, using a Gilford Model 2410-S linear transport attachment. Two bands appear, the band from the cathode side is cut out as a 2 mm slice and several such slices are mixed with glass beads (Superbrite glass beads, type 130-5005, Reflective Product Division 3 M Co., Minnesota; about one quarter of the volume of the slices is used) and ground with a glass rod. Phosphate buffer, 0.01 M, pH 6.0, is then added; the suspension is well mixed and filtered through a fine sintered-glass filter, and more of the buffer is used for washing (total volume of 8 ml per six slices). The filtrate is dialyzed against 0.05 M borate buffer, pH 8.17 (three changes of 50 ml at 4°; the dialysis bags pretreated by heating them at 100 ° for 20 min in 0.01 M EDTA, are washed with double-distilled water and stored at 4 °) and concentrated by ultrafiltration. One run with six gels yields 0.26 mg 11 of the pure enzyme. A. Tiselius, S. Hjerten, and O. Levin, Arch. Biochem. Biophys. 65, 132 (1956).

1oB. J. Davis, Ann. N.Y. Acad. Sci. 121,404 (1964). 110. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951).

[50]

DIPEPTIDYL CARBOXYPEPTIDASE

607

Stability. When stored at --20 ° this preparation retained 70% of the original activity after 5 weeks. It is advantageous to keep stored the preparations obtained by hydroxyapatite chromatography in the previous step. Even when loss of activity occurs, pure preparations, having the highest specific activity (110-120 units/rag) can be recovered by electrophoresis, but with an accordingly lower yield.

P u r i t y and Properties

Polyacrylamide Gel Electrophoresis. The enzyme migrates as a single component when subjected to polyaerylamide gel electrophoresis in 7.5% gels at pH 8.3 and 7.0 by the method of Ornstein and Davis, L° in gels of graded porosity, 13 and when the denatured enzyme is subjected to gel eleetrophoresis in presence of sodium dodeeyl sulfate) 4 Immunodiffusion and Immunoeleetrophoresis. Rabbits were immunized with a partially purified enzyme, and the serum was used to establish the homogeneity of D C P by immunodiffusion and by immunoelectrophoresis. A single preeipitin arc is observed with the pure DCP, whereas a nmnber of arcs are seen with a D C P preparation obtained after the gel-filtration step. Molec~dar Weight. The molecular weight of the native D C P was estimated as 97,000 by eleetrophoresis through polyacrylamide gels of graded porosity, ~ using ovalbumin (MW 45,000) and bovine serum albumin (MW 68,000 for monomer, 138,000 for dimer) as the markers. pH Profile. The dependence of D C P activity on pH is represented by a bell-shaped curve with a maximum at pH 8.2. This was shown with Dnp(Pro-Gly-Pro),~ as the substrate, in 0.05 M Veronal buffer, in the pH range from 7.0 to 9.0. Metal Ion Req~drement. The enzyme, as obtained, is active without the addition of metal ions. Exhaustive dialysis against 0.1 m M E D T A does not affect the specific activity. Addition of Mg 2÷, 5in -~÷, Zn 2+, Cd 2÷, and Ni '-'÷ has a very small effect, but the addition of Co 2÷ (1 ~M to 1 raM) increases the activity about 5-8 times, with a maximal effect at 50 t~M. Z-Ala3 was used in these experiments as the substrate, and the release of alanylalanine was followed by the colorimetrie ninhydrin method of Rosend 1.~L. Ornstein and B. J. Davis, Ann. N.Y. Acad. Sci. 121,428 (1964). 1~L. Kamm and J. Mes, d. Chromatogr. 62, 383 (1971). ~A. L. Shapira, E. Vifiuela, and J. V. Maizel, Biochem. Biophys. Res. Commun. 28, 1815 (1967). 15 L . O. Anderson, H. Borg, and M. Mikaelsson, FEBS Lett. 20, 199 (1972).

608

EXOPEPTIDASES

[50]

Substrate Specificity. DCP hydrolyzes the penultimate peptide bond of a-N-blocked tripeptides, free tetrapeptides, and higher peptides. The following compounds are typical examples: Z-Ala-Ala-Ala, GIy-Gly-Phe-Ala,

Ala-Ala-Ala-Ala, Lys-Lys-Lys-Lys,

Boc-Ala-Glu-Ala-Ala, Ala-Ala-Ala-Lys-Phe,

Lys-Ala-Ala-Lys-Ala-Ala, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (0.05 M borate buffer, pH 8.1, 75 ~M COSO4, 40 °, 2 t~g/ml enzyme). Free tripeptides are not hydrolyzed, therefore N-protected tripeptides are the smallest substrates for DCP. A free carboxyl group is required, since no hydrolysis occurs with tetraalanine amide. Secondary peptide bonds to which the nitrogen is donated by a proline residue, as in Z-Phe-Pro-Ala or polyproline, are not cleaved. Neither is a peptide consisting of a chain of several glycine residues, such as Z-Gly-GlyGly-Gly. Peptides with a C-terminal D-residue are resistant to DCP. Since DCP acts on the carboxyl end of polypeptides by hydrolyzing successively every second peptide bond, one may expect to find it useful for application in studies of protein structure.

Kinetic Parameters Dependence of Specific Activity on Enzyme Concentration. Linear increase of activity with increasing enzyme concentration was found up to a DCP concentration of 1.5 t~g/ml with Boc-Ala3 as the substrate (5raM in 50 mM borate buffer, pH 8.15, at 40°). At higher concentration, the dependence deviated from linearity, the specific activity becoming smaller with increasing enzyme concentration. Thus, while the rate of hydrolysis per 1 ~g of enzyme per milliliter was 8 nmoles min -1 in the linear range, it was 7 nmoles rain -1 at an enzyme concentration of 2 t~g/ml and only 6 nmoles rain -~ at an enzyme concentration of 3 ~g/ml. Michaelis-Menten Parameters. The kinetic parameters, Km and kent, for several tri- and tetrapeptides are given in Table II. Initial rates were obtained by the pH stat method or spectrophotometrically by measuring the absorption at 225 nm. The kinetic constants were calculated from Lineweaver-Burk plots that were linear in the substrate concentration range of 0.1-7.5 mM. The enzyme shows high affinity for basic residues as can be seen by the substrate inhibition observed for Lys4 and the pronounced product inhibition with lysyl and arginyl dipeptides.

[50]

D I P E P T I D Y L CARBOXYPEPTIDASE

609

TABLE II KINETIC PARAMETEI~S FOR THE HYDROLYSIS OF PEPTIDES BY I)IPEPTIDYL CARBOXYP EI'TIDASE

Substrate or competitive inhibitor

Ko ~ (m M)

Ki c (uM)

k¢~t (see-l)

Substrate cone. range (raM)

Boc-Alaa Ala4 S-Ala4 Ala-Ala-Phe-Ala Gly-Ala-Phe-Ala Ala-Gly-Phe-Ala Gly-Gly-Phe A l a Lys4 Ala2 Lys-Ala Ala-Lys Phe-Arg

0.71 0.44 0.41 1.27 1.39 0.61 6.06 ~ 0 . 1 0 l' -----

--------396 19.3 9.0 0.92

34 139 131 225 194 156 116 "~30 I' -----

0.8-5.0 0.6-5.0 0.5-5.0 1.0-5.0 1.0-5.0 1.0-5.0 1.0-5.0 O. 0 8 - 1 . 0 -----

a Km values were determined spectrophotometrically. (For Z-Ala4 the p H - s t a t m e t h o d was also used, resulting in identical Km value.) The composition of the reaction mixture was: substrate in 0.05 M borate buffer, p H 8.15 (no metal added) at 40 °, enzyme (1.0 to 6 nM). The difference in molar extinction coefficients, Ae225 accompanying the hydrolysis was determined with authentic mixtures of the product peptides and the substrates. In the p H - s t a t method, the substrate and enzyme in 0.1 M KC1 were kept at p H 8.15 with 0.002 M KOH, at 40 °. The reaction mixture volume was 1 ml. b Substrate inhibition. c Competitive inhibition, p H - s t a t method used with Z-Ala4 as the substratc (A. Yaron and D. Mlynar, unpublished results). Occurrence In its specificity requirements DCP resembles the "angiotensin Iconverting enzyme" present in mammalian tissue. This "converting enz y m e , " f i r s t d i s c o v e r e d b y S k e g g s e t al. TM i n h o r s e p l a s m a w a s s h o w n t o be a dipeptidyl earboxypeptidase or peptidyldipeptide hydrolase (EC 3.4.15.1), previously also termed kininase IIY It thus appears that d i p e p t i d y l c a r b o x y p e p t i d a s e b e l o n g s t o a f a m i l y of e n z y m e s o c c u r i n g b o t h i n b a c t e r i a l a n d m a m m a l i a n o r g a n i s m s . I t is of i n t e r e s t t h a t d u r i n g the various purification steps, DCP was accompanied by a dipeptidase, an enzyme that specifically hydrolyzes dipeptides, which are the prod26L. T. Skeggs, W. H. Marsh, J. R. Kahn, and N. P. Shumway, J. Exp. Med. 99, 275 (1954). 17 H. Y. T. Yang, E. G. Erd5s, and Y. Levin, Biochim. Biophys. Acta 214, 374 (1970).

610

EXOPEPTIDASES

[51]

ucts of D C P action. The two enzymes were not separated by ion-exchange column chromatography on DEAE-cellulose, by gel filtration, or by polyacrylamide gel electrophoresis. Even the a c t i v i t y - p H curves were found to be very similar. Separation of the two enzymes was eventually achieved by chromatography through hydroxyapatite columns. The complementary action of the two enzymes on the carboxyl ends of polypeptide chains represents an alternative mechanism for carboxypeptidase action by which a polypeptide chain is successively degraded from the carboxyl end to amino acids. An interesting regulatory mechanism is indicated by the finding that the dipeptides formed by D C P act as competitive inhibitors of this enzyme and in turn serve as substrafes for dipeptidase, the activity of which depends on Mn ions. Acknowledgment The financial support granted by the Helena Rubinstcin Foundation Inc. is gratefully acknowledged.

[51] E x o c e l l u l a r D D - C a r b o x y p e p t i d a s e s - T r a n s p e p t i d a s e s from Streptomyces B y JEAN-MARIE FR~RE, M~LINA LEYH-BouILLE, JEAN-MARIE GHUYSEN,

MANUEL I~IETO, and HAROLDR. PERKINS Strains and Culture Strains. Strains R39 and R61 are soil isolates2,-" Their designations are arbitrary. In strain R39, the cross-link between the peptide units of the wall peptidoglycan extends from the C-terminal D-alanine of one unit to the amino group at the D-center of meso-diaminopimelic acid of another unit 3 (peptidoglycan of chemotype I).~ The interpeptide bond is in ~-position to a free carboxyl group. In strain R61, the cross-link extends from a C-terminal D-alanine of a peptide unit to a glycine residue attached to the ~-amino group of LL-diaminopimelic acid of another peptide unit 5 (peptidoglycan of chemotype II). 4 The exocellular DD-

i M. Welsch, Rev. Belge Pathol. Med. Exp. 18, Suppl. 2, 1 (1947). 2M. Welsch and A. Rutten-Pinckaers, Bull. Soc. R. Sci. Liege, 3-4, 374 (1963). J.-M. Ghuysen, M. Leyh-Bouille, J. N. Compbell, R. Moreno, J.-M. Fr~re, C. Duez, M. Nieto, and H. R. Perkins, Biochemistry 12, 1243 (1973). J.-M. Ghuysen, Bacteriol. Rev. 32, 425 (1968). M. Leyh-Bouille, R. Bonaly, J.-M. Ghuysen, R. Tinelli, and D. J. Tipper, B/ochemistry 9, 2944 (1970). 4