JOURNAL OF BACTERIOLOGYVol. 88, No. 3, p. 653-659 September, 1964Copyright ( 1964 American Society for Microbiology

Printed in U.S.A.

PROPERTIES OF PROTEINASE FROM STREPTOCOCCUSFAECALIS VAR. LIQUEFACIENS'

ARNOLD S. BLEIWEIS2 AND LEONARD N. ZIMMERMAN

Department of Microbiology, The Pennsylvania State University, University Park, Pennsylvania

Received for publication 29 April 1964

ABSTRACT

BLEIWEIS, ARNOLD S. (The Pennsylvania StateUniversity, University Park), AND LEONARD N.ZIMMERMAN. Properties of proteinase from Strep-tococcus faecalis var. liquefaciens. J. Bacteriol. 88:653-659. 1964.-The extracellular group D strepto-coccal proteinase is inactivated by chelatingagents [ethylenediamine-tetraacetate (EDTA), o-phenanthroline, and 8-quinolinol] and mercaptans(cysteine, mercaptoethanol, and thioglycolate).The optimal inhibitory concentrations of EDTA(4 X 1O4 M) and cysteine (2.5 X 1O2 M) promoterapid loss of activity with 50% inactivation after4 to 5 min. Enzyme inactivated by either EDTA orcysteine is reactivated about 80% by 4 X 10-4 MZn++. Such reactivation of EDTA-treated enzymeis prevented completely by iodoacetate (5 X 10-M) and of cysteine-treated enzyme by oxidizingconditions, which suggests that the zinc binding-site may be a thiol. High levels of zinc (10-3 M) donot allow reactivation in either case, and actuallyinhibit native proteinase. Ca++, Mg++, Co++, Fe++,Cu++, and Nil do not reactivate cysteine-treatedenzyme, but Mn (10-4 to 8 X 104 M) allows 27%reversal. N2-held, cysteine-treated enzyme can bespontaneously reactivated if the substrate isflushed with O0 during the assay; leakage of air or02 into the samples before assay leads to loss ofreactivatability. Native proteinase does not loseactivity after dialysis for 43 hr against 0.07 Mphosphate buffer. It is concluded that the group Dproteinase obtained from Streptococcus faecalisvar. liquefaciens is probably a zinc metalloenzymethat is unlike the thiol -activated, group A strepto-coccal proteinase, but similar to the mammaliancarboxypeptidase A.

Group A streptococci elaborate an extracellularproteinase (Elliott, 1945) as a zymogen (Elliott

1 Journal paper no. 2801, Pennsylvania Agri-cultural Experiment Station. Part of a Ph.D.Thesis submitted by the senior author to theGraduate School of the Pennsylvania State Uni-versity.

I Present address: Department of PreventiveMedicine and Public Health, Washington Uni-versity School of Medicine, St. Louis, Mo.

and Dole, 1947; Elliott, 1950) which is activatedby both proteolysis and exposure to reducingagents. This proteolytic activation can be accom-plished autocatalytically in the culture medium,or by trypsin. With the use of crystallized pre-cursor and the active proteinase, the serologicaland electrophoretic (Shedlovsky and Elliott,1951) properties of each form were determinedand, upon treatment of the crystalline zymogenwith trypsin or reducing agents, products dem-onstrating the appropriate proteinase propertieswere obtained. The enzyme resulting from tryp-sinization is not fully active, however, unlessmercaptans or cyanide are added, and once ac-tivated the enzyme is irreversibly inactivated byiodoacetate (Elliott, 1950). Recently (Liu et al.,1963), the chemical properties of the two proteinswere determined more fully through amino acidanalyses, end-group determinations, and molecu-lar weight studies of chromatographically purifiedsamples.Group D streptococci also produce an extra-

cellular proteinase. The enzyme was partiallypurified by Grutter and Zimmerman (1955), themetabolic optima for enzyme biosynthesis weredetermined (Rabin and Zimmerman, 1956; Hart-man, Zimmerman, and Rabin, 1957), and bio-synthetic control mechanisms were studied(Hartman and Zimmerman, 1960). The presentcommunication details initial chemical studies ofthe enzyme, and presents data that indicate basicdissimilarities with the group A proteinase.

MATERIALS AND METHODS

Organism. Streptococcus faecalis var. liquefa-ciens, strain 31, was used as a source of proteinase.(The original stock was obtained from the Divi-sion of Bacteriology, Cornell University.) It wascultured always at 37 C.

Media. A-C broth (Rabin and Zimmerman,1956) was used to bring up the inoculum for theinduction medium. The latter, N-Z Case syn-thetic medium, was constituted as follows:Sheffield N-Z Case, 0.2 g; L-arginine HCl, 0.193

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g; lactose, 0.2 g; KH2PO4 1.0 g; NaCl, 0.2 g;MgSO4-7H20, 8 mg; FeSO4-7H20, 0.4 mg;MnSO4'H20, 0.16 mg; CoCl2, 0.12 mg; CaCl2,1.5 mg; adenine, guanine, and uracil, 0.5 mg each;riboflavine, Ca pantothenate, pyridoxine -HCl,and niacin, 0.1 mg each; folic acid, 1 ,g; biotin,0.1 ,ug; and distilled water to 100 ml. The mediumwas adjusted to pH 7.0.

Proteinase induction. A 1% washed inoculumof cells previously grown for 12 hr in A-C brothwas added to 1 liter of N-Z Case synthetic me-dium. After 6 hr of incubation, 10 ml of a sterilearginine-lactose solution were added. (Theamounts of arginine and lactose in the solutionwere equal to those used for 1 liter of the originalmedium, and constituted a nutritional "booster"to increase total enzyme biosynthesis.) The cul-ture was reincubated for another 4 hr, at whichtime the cells were removed by centrifugation at14,500 X g for 5 min.

Proteinase purification. A modification of themethod of Grutter and Zimmerman (1955) wasemployed to recover the proteinase. (This pro-cedural change was necessary because of thechange of induction media; however, the degreeof recovery and purity of enzyme was similar tothat reported in the original paper.) Enough solid(NH4)2SO4 was added slowly to the clarified cul-ture supernatant fluid to allow 0.8 saturation(0 C). After 40 hr at 8 C, the resulting precipitatewas collected by centrifugation at 14,500 X g for1 hr. This was dissolved in 20 ml of cold 0.15saturated (0 C) (NH4)2SO4, previously adjustedto pH 8.3, and was immediately centrifuged at15,300 X g for 15 min to remove insoluble ma-terial. In the resulting supernatant fluid (adjustedto pH 6.3 with 6 N HCl) was suspended a cello-phane bag containing 20 ml of 1.0 saturated(O C) (NH4)2SO4. The supernatant fluid was heldthus at 8 C for 14 hr, at which point the newlyformed precipitate outside the bag was collectedby centrifugation at 35,000 X g for 15 min, dis-solved in 20 ml of 0.07 M phosphate buffer (pH7.0), and centrifuged at 15,300 X g for 15 minagain to remove insoluble material. The clarifiedsupernatant fluid was divided into 1-ml portionswhich were frozen at -17 C in individual cap-sules.

Protein determination. Protein concentrationwas determined by the biuret reaction (Gornall,Bardawill, and David, 1949), with a 0.4% solution

of bovine plasma 'y-globulin (Armour and Co.,Chicago, Ill.) used as the standard.

Proteolytic activity determination. Enzymatic ac-tivity was measured by the method of Anson(1938). In the present studies, 1.0 ml of enzymesample was added to 5.0 ml of 1% vitamin-freecasein solution, prepared as described previously(Grutter and Zimmerman, 1955). After 1 hr at37 C, 10.0 ml of 0.6 M trichloroacetic acid wereadded, and the precipitate was removed by fil-tration through Whatman no. 4 filter paper. Theblank for each sample was prepared by adding1.0 ml of enzyme to 10.0 ml of trichloroacetic acidplus 5.0 ml of casein substrate and filtering im-mediately. Solubilized aromatic amino acids weredetermined by measuring the absorbancy of atrichloroacetic acid filtrate at 280 m,u with aBeckman model DU spectrophotometer. A stand-ard curve for tyrosine allowed conversion ofabsorbancy units to "micrograms of solubilizedtyrosine per 6 ml."

In experiments in which an oxygenated sub-strate was employed, the procedure was as fol-lows: 5.0 ml of casein substrate were preflushedwith 02 for 10 min at 37 C in side-arm test tubes.At zero time, any lost volume was restored withdistilled water; 1.0 ml of sample was addedquickly, and 02-flushing continued for 30 min. At30 min, the tubes were clamped off and were heldfor the final 30 min. At 60 min, the volume wasrestored with water, and trichloroacetic acid wasadded.

Dilution of proteinase stock for routine experi-ments. Before each experiment, a capsule of en-zyme was thawed and was then diluted with either0.07 M potassium phosphate buffer or distilledwater. The dilutions were arranged so that astandard control activity was obtained for all ex-periments. This final activity was 1,200 ,ug ofsolubilized tyrosine per 6 ml + 10%. Such dilu-tions resulted in protein concentrations of0.0023 mg/ml L 0.0007 mg.

Chemicals. Iodoacetic acid, Na thioglycolate,2-mercaptoethanol, 8-quinolinol, and o-phen-anthroline were obtained from Eastman ChemicalProducts, Inc., Kingsport, Tenn.; the cysteine.HCl and ethylenediamine-tetraacetic acid(EDTA) were obtained from Mann ResearchLaboratory, New York, N.Y. and from AmendDrug and Chemical Co., Inc., New York, N.Y.,respectively.

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VOL. 88, 1964 PROTEINASE FROM S. FAECALIS VAR. LIQUEFACIENS 6.55

RESULTS

Effects of reducing agents. Reducing agents, 140rather than acting in a stimulatory fashion as is 120C Athe case for group A streptococcal proteinase, ac-tually inhibit group D proteinase (Table 1). \Cysteine and 2-mercaptoethanol were more effec- 800\tive than thioglycolate; but, with increased |600\concentration and incubation time, even the \latter showed total inhibition. Cysteine, however, 2was chosen for further study, and the optimal X 200inhibitory concentration was found to be 2.7 X .0210-2 M, as indicated by the extrapolation of curve CYSTONE CONCENTRATON(M)A, Fig. 1. (In all subsequent experiments, 2.5 X FIG. 1. Effects on enzymatic activity of preincu-10-2 M was used and provided 90 to 100%0/0 inhibi- bation with various cysteine concentrations. Thetion regularly.) A slight (11%) stimulation by reaction mixtures contained standard enzyme con-10-3 M cysteine could be noticed, but thereafter centrations and cysteine as indicated in 0.07 M phos-increasing cysteine produced inhibition. To lessen phate buffer (pH 6.5) at a volume of 4.0 ml. Incuba-the degree of cysteine degradation by air, the ex- tion, before assay, was for 1.5 hr at 37 C in air (A)periment was repeated under N2. Curve B (Fig. 1) or under N2 (B).indicates, surprisingly, that the inhibitory effi-ciency of the mercaptan is decreased under these 10anaerobic conditions, and that the optimalinhibitory concentration is increased to 3.6 X10-2 M. The rate of cysteine inhibition of pro- 90_teolytic activity (in air) was studied to determinethe optimal time for total inactivation. Fig. 2 80sshows a rapid loss of activity, with 50% inactiv-ation after 4 min and 90% after 20 min. 70.

Effects of chelating agents. EDTA and o-phenan-throline completely inactivate group D protein-ase, whereas 8-quinolinol is somewhat less effec- 60 /tive (possibly due to limited solubility in the

TABLE 1. Effects of three reducing agents on groupD proteinase* 40IIncuba- Inhibi-

Mercaptan Concn tion time tionat 37 C

M hr %

Sodium thioglycolate. 0.1 1 41.0 1 520.1 2 201.0 2 100

2-Mercaptoethanol ....... 0.1 1 261.0 1 86

Cysteine................. 0.1 2 971.0 2 95

* The reaction mixtures contained enzyme (seeMaterials and Methods for standard concentra-tion) and mercaptans as indicated in 0.07 Mphosphate buffer (pH 6.5) at a volume of 2.0 ml.

T1ME (min)

FIG. 2. Rate of cysteine-inhibition of proteolyticactivity in air. Standardized enzyme and cysteine(2.5 X 1-2 M) in 0.07 M phosphate buffer (pH 7.0)at a volume of 56 ml were incubated at 37 C. At regu-lar intervals, 1.0-ml portions were withdrawn forassay.

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buffer), and citrate is totally without effect (Ta-ble 2). To preclude the possibility that the EDTAwas affecting the casein (and thus the enzymeonly indirectly), 102 M EDTA and casein sub-strate (5.0 ml) were incubated at 37 C for 1 hrbefore assay with 1.0 ml of untreated enzyme.Only 24% inhibition was observed, as comparedwith 94% inhibition when the enzyme was pre-incubated with EDTA (Table 2). The optimalconcentration of EDTA for total enzymatic in-hibition was found to be 3.9 X 10-4 M whenenzyme and chelator, at several concentrations,were mixed in 0.07 M phosphate buffer (pH 7.0)at a volume of 4.0 ml and held at 37 C for 1 hrbefore assay. The rate of enzymatic inhibition byEDTA was determined under the same condi-tions as those for cysteine inhibition (Fig. 2). Thecurve (not shown) was similar to that in Fig. 2,revealing a rapid loss of activity with 50%inhibition in 5 min and 90% in 25 rin.

Reactivation by metallic ions. Various divalentmetallic ions of the first transition series areknown to act as enzyme cofactors. Some of these(Ca++, Mg++, Fe+, Co+, Ni++, Cu+, Mn ,and Zn++), as the sulfate salt, were tested forreactivation of cysteine-inactivated proteinase.To the main compartments of double side-armWarburg flasks were added 3.0 ml of enzyme; tothe stoppered side arms, 0.5 ml of cysteine; andto the vented side arms, 0.5 ml of metallic-ionsolution. The diluent for all components wasdistilled water, because insoluble metallic phos-phates are formed in phosphate buffer. Uponcompletion of mixing, the 4.0-ml samples werecomposed of enzyme at the standard concentra-tion, cysteine at 2.5 X 102 M, and one of theabove metallic ions at either 6.3 X 10-3 M, 1.6 X10r3 M, 3.9 X 10- M, or 9.9 X 10-5 M. Incu-bations were at 37 C.

TABLE 2. Effects of four chelating agentson group D proteinase*

Chelator Inhibition

Ethylenediamine-tetraacetate ........ 948-Quinolinol .......................... 40o-Phenanthroline.................... 100Sodium citrate...................... 0

* Standardized enzyme and 10-2 M chelator asindicated were reacted in 0.07 M phosphate buffer(pH 7.0) at a volume of 5.0 ml. Incubation, be-fore assay, was at 37 C for 1 hr.

z0

0

ma

B

2pM

FIG. 3. Reactivation with (A) Zn++ and (B) Mn+of cysteine-inactivated proteinase. The complete re-action mixture consisted of standardized enzyme,cysteine (p.5 X 10-2 M), and either Zn or Mn++ asindicated. Incubation was at 87 C. (See text forprocedural details and definition of pM.)

The vessels were flushed with N2 for 15 minbefore the cysteine was tipped into the enzyme.N-flusbing was continued for an additional 20min, at which time gas vents were closed and themetallic ion solutions were added to the cysteine-treated enzy me. Mixing, under N2, was allowedto proceed for 1 hr. At that point, all vents wereopened to air, and the samples were shaken foranother 1.5 hr before assay.

It was found that Ca++, Mg++, Co++, Nil ,Cu++, and Fe++ do not reactivate, but Mn++ isslightly effective and Zn- allows almost com-plete regeneration of enzymatic activity. Figure3, in which the reactivation is plotted as a func-tion of the negative log of the metallic-ionconcentration (pM) (Vallee, 1960), demonstratesthe effects obtained with (A) Zn+ and (B) Mn .

Zn++ reactivation is maximal (81%) at 3.9 X10-4 M and, in the case of Mn-l, 10-4 to 8 X10-4 M is the approximate optimal range (27%reversal).

In each case, however, superoptimal metal con-centrations reactivate to a lesser extent than dothe optimal concentrations. This effect is more

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pronounced with Zn , where the highest levelsdo not allow any reactivation. This indicates thatZn++ actually might be inhibitory at these highlevels, although lower amounts are necessary foractivity. To test this hypothesis, native protein-ase was incubated at 37 C for 1 hr with variousZn++ concentrations before assay. Enzyme wasinactivated linearly as a function of increasingmetallic-ion concentration above 7.9 X 10-4 M.At 6.3 X 10-3 M, there was 90% reduction ofactivity.

Znt1 reactivation of cysteine-inactivated pro-teinase did not occur in air. If air was introducedinto the Warburg flasks after cysteine was tipped,but before Zn++ was added, there was no reac-tivation. Once the metal was added, however, thesystems could be opened and activity could bedetermined. Although the samples were routinelyincubated for 1 hr after addition of Zn++ andbefore aeration, opening the systems after only 5min did not prevent reactivation.To determine whether EDTA-inactivated en-

zyme also could be reactivated by Zn , an ex-periment was performed identical to thatdescribed above for reactivation of cysteine-treated proteinase. EDTA replaced cysteine inthe stoppered side arm, but all other componentsand conditions were as before.

It was found that the enzyme was reactivatedto the extent of 83% by 3.9 X 10-4 M Znl+.Other concentrations above (6.3 x 10-3 M and1.6 X 10-3 M) and below (9.9 x 10-5 M and2.5 X 10-5 M) the optimum did not exhibitproteolytic activity.

Interestingly, anaerobic conditions are not re-

quired for reactivation of EDTA-treated pro-teinase. The previous experiment (with the use

of only 3.9 X 10-4 M Zn ) was repeated in airwithout N2-flushing. Reactivation to the extentof 73% resulted.

Effect of iodoacetate on Zn++ reactivation. Thethiol poison, iodoacetate, does not significantlyaffect native group D proteinase (Rabin andZimmerman, 1956). The effect of the poison on

Zn reactivation of EDTA-inactivated enzymewas studied in the following manner. In test tubes,standardized enzyme and optimal EDTA forinactivation were incubated at 37 C for 30 min(at a volume of 3.5 ml). To one tube was added5.0 X 10-2 M iodoacetate (0.5 ml) and to theother, distilled water (0.5 ml). Incubation at 37 Cwas continued for 60 min when 3.9 X 10-4 M

Zn++ (0.5 ml) was added to each sample. Afterfurther incubation for 60 min, they were assayed.The diluent for each component was water.As expected, the addition of water and Zn+

allowed 82% reactivation, but no reactivationwas obtained from the sample pretreated withiodoacetate before addition of the metallic ion.Spontaneous reactivation in oxygenated sub-

strate. Proteinase, treated with cysteine underN2, can be reactivated spontaneously if samplesare assayed in a casein substrate that is beingflushed (not bubbled) with 2 . To the main com-partments of double side-arm Warburg flaskswere added 3.0 ml of enzyme; to the stopperedside arms, 0.5 ml of cysteine; and to the ventedside arms, 0.5 ml of 0.07 M phosphate buffer (pH7.0). The diluent was 0.07 M phosphate buffer(pH 7.0), and incubation was at 37 C. The 4.0-mlfinal reaction mixture contained the standardenzyme concentration and 2.5 X 10-2 M cysteine.The vessels were flushed with N2 for 15 min

before both side arms were tipped into the en-zyme. The reaction mixtures were then incubatedfor 20 min under N2 before immediate assay eitheras usual or with substrate being flushed rapidlywith 02-

Samples assayed as usual (i.e., with nonoxy-genated substrate) indicated approximately 50%inactivation compared with 90 to 100% inactiva-tion obtained when both cysteine treatment andassay were carried out in the air. Samples assayedin oxygenated substrate showed only 15% inac-tivation in contrast to 85% inhibition obtainedwhen cysteine treatment was in air and assay wasunder 02-The sensitivity of cysteine-treated proteinase

to irreversible inactivation by air was demon-strated by opening the Warburg flasks after thenormal 20-min incubation under N2, and assay-ing enzyme samples shaken for varying lengths oftime in air. Increased exposure to air caused alinear decay in the ability to be reactivated byoxygenated substrate. By 36 min no reactivationwas possible. When 02 was flushed through theWarburg flasks, after 20 min under N2, an evensteeper decay curve (not shown) was obtained,with the end point at 10 min.

Proteinase, treated with EDTA under N2,was not reactivated in oxygenated substratewhen the experiment described above was re-

peated with the chelating agent replacing themercaptan.

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Dialysis of native proteinase. When native groupD proteinase was dialyzed against 0.07 M phos-phate buffer (pH 7.0) for 43 hr, no loss of activityresulted. Enzyme (10.0 ml), at the standard con-centration, was added to a dialysis bag whichwas then suspended in 100 volumes of buffer. Thebag was held at 8 C and the buffer was changedevery 12 to 14 hr. After 43 hr, the enzyme wasassayed and compared with an undialyzed, con-trol isample.

DISCUSSIONThe chemical studies of the group D strepto-

coccal proteinase indicate that the enzyme maybe a zinc metalloenzyme. Loss of proteolytic ac-tivity occurs when the enzyme is preincubatedwith the chelating agents, EDTA, 8-quinolinol,and o-phenanthroline (Table 2). It is worth not-ing, however, that citrate may be noninhibitory,because this chelating agent apparently does notbind Zn++ or Mnt (Hodgman, 1960).Although the inhibitory effects of mercaptans

might be due to their reducing abilities, it appearsthat chelation is the mechanism involved here.Reactivation in this system is accomplished byZn++ at the same optimal level as for reactivationof EDTA-treated enzyme (Fig. 3). Except forMn++, which allowed slight reversal, all otherdivalent metallic ions tried were without effect,demonstrating a definite metal specificity. Theinteresting aspect of metal reactivation of cys-teine-treated proteinase is the absolute require-ment for anaerobic conditions. Whereas EDTA-treated enzyme is reactivated both in air andunder N2, cysteine-treated enzyme is irreversiblyinactivated in air, and can only be reactivatedwhen treatment is under N2. It seems that al-though both cysteine and EDTA inhibit throughchelation, which is reversible primarily by Zn++in each case, the mercaptan has a secondaryinhibitory mechanism, which requires 02 and isirreversible by the metal. This oxidative inhibi-tion may involve addition of cysteine to the zincbinding-site, thereby blocking readdition of themetal. The binding-site for zinc on proteins wasfound to be sulfur atoms (Williams, 1959), as inthe case of carboxypeptidase A (Coombs, Felber,and Vallee, 1962). Such enzymes are usually in-activated by iodoacetate which carboxymethyl-ates thiol groups, including those acting as zincbinding-sites. Native group D proteinase is notaffected by iodoacetate (Rabin and Zimmerman,

1956), but EDTA-inactivated proteinase that istreated with the thiol poison cannot be reacti-vated. It appears, therefore, that chelation of theenzyme involves the exposure of such a suscepti-ble site.The decreased amounts of inhibition produced

by cysteine under N2 (Fig. 1, curve B) can beexplained by the above hypothesis. In addition,a mechanism for spontaneous reactivation inoxygenated casein substrate can be presented thatinvolves (i) protection of chelated enzyme firstby N2 and then by substrate from irreversibleinactivation, and (ii) elimination of cysteine bymeans of oxidation to cystine. This latter reactionwould free zinc formerly bound as zinc cysteinate.But, if air or O2 gets into the N2-held samplebefore it is added to substrate, irreversible inac-tivation occurs, owing to cysteination of the thiol,zinc binding-site.

Interestingly, not only a metal specificity wasdemonstrated but also a concentration specificitywas shown. Superoptimal zinc concentrations failto reactivate chelator- or mercaptan-treated en-zyme (Fig. 3), and also inhibit native enzyme.This unusual phenomenon also was observed withthe zinc metalloenzyme carboxypeptidase A,which likewise is inactivated by zinc at 10-3 Mand above (Vallee et al., 1960). This, however, isnot the only resemblance between enterococcalproteinase and mammalian carboxypeptidase A.Coombs et al. (1962) reported the inhibition ofthe latter enzyme by several chelating agents,including o-phenanthroline. Inhibition is a directfunction of zinc removal, indicating the probablepresence of the metal at the active site. Cysteine,mercaptoethanol, and thioglycolate inactivatecarboxypeptidase by the same mechanism. Theseinvestigators suggest that the reason for theextraordinary effectiveness of these reducingagents, acting as chelators, is very likely that zincis bound to the enzyme at a sulfur-nitrogen site(Coleman and Vallee, 1961) and the mercaptanscompete for the metal. Inhibition of carboxypep-tidase by mercaptans is a function of both theirconcentration and time of preincubation withenzyme. Table 1 suggests a similar function withthe enzyme presently under consideration. Thedifferential effectiveness of these inhibitors maybe due to differences in the stability constants ofthe zinc-mercaptan complexes (Coombs et al.,1962).

Inactive carboxypeptidase, treated with o-

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phenanthroline, can be fully reactivated by addi-tion qf zinc ions equimolar to the original chelatorconcentration (Felber, Coombs, and Vallee, 1962).Similarly, enterococcal proteinase, which is inac-tivated optimally by approximately 4 X 10-4 MEDTA, can be reactivated almost completely byaddition of the same concentration of Zn++. Car-boxypeptidase is also reactivated by Co++, Ni+,Fe-++, and Mn+ (Coleman and Vallee, 1961),although the zinc enzyme is the most stable form(Coombs et al., 1962). The streptococcal enzyme,however, is only affected by Zn++ and Mn .

There is, therefore, some definite resemblancebetween the two enzymes, although the chemicaldetermination of zinc bound to the streptococcalproteinase is the ultimate proof of its status as a

metalloenzyme. If such zinc does indeed exist, itis bound very firmly to the proteinase molecule,because dialysis for 43 hr against 0.07 M phos-phate buffer failed to reduce enzymatic activity.On the other hand, there is little resemblance tothe group A proteinase shown by the group Denzyme. Whereas the former is activated by mer-

captans and is unaffected by 10-3 M EDTA (Liuet al., 1963), the latter is inactivated by each.Also, the group A enzyme is inactivated by iodo-acetate (Elliott, 1950), whereas the enterococcalenzyme is unaffected by the poison (Rabin andZimmerman, 1956).

ACKNOWLEDGMENT

This investigation was supported in part bygrant GB1399 from the National Science Founda-tion.

LITERATURE CITED

ANSON, M. L. 1938. The estimation of pepsin,trypsin, papain, and cathepsin with hemo-globin. J. Gen. Physiol. 22:79-89.

COLEMAN, J. E., AND B. L. VALLEE. 1961. Metallo-carboxy-peptidases: stability constants andenzymatic characteristics. J. Biol. Chem. 236:2244-2249.

CooMBs, T. L., J.-P. FELBER, AND B. L. VALLEE.1962. Metallocarboxypeptidases: mechanismof inhibition by chelating agents, mercaptans,and metal ions. Biochemistry 1:899-905.

ELLIOTT, S. D. 1945. A proteolytic enzyme pro-duced by group A streptococci with specialreference to its effect on the type-specific Mantigen. J. Exptl. Med. 81:573-592.

ELLIOTT, S. D. 1950. The crystallization and sero-

logical differentiation of a streptococcal pro-teinase and its precursor. J. Exptl. Med. 92:201-218.

ELLIOTT, S. D., AND V. P. DOLE. 1947. An inactiveprecursor of streptococcal proteinase. J.Exptl. Med. 85:305-320.

FELBER, J.-P., T. L. COOMBS, AND B. L. VALLEE.1962. The mechanism of inhibition of carboxy-peptidase A by 1, 10-phenanthroline. Bio-chemistry 1:231-238.

GORNALL, A. G., C. J. BARDAWILL, AND M. W.DAVID. 1949. Determination of serum proteinsby means of the biuret reaction. J. Biol. Chem.177:751-766.

GRUTTER, F. H., AND L. N. ZIMMERMAN. 1955. Aproteolytic enzyme of Streptococcus zymo-genes. J. Bacteriol. 69:728-732.

HARTMAN, R. E., AND L. N. ZIMMERMAN. 1960.Effect of the arginine dihydrolase enzyme sys-tem on protein biosynthesis by Streptococcusfaecalis var. liquefaciens. J. Bacteriol. 80:753-761.

HARTMAN, R. E., L. N. ZIMMERMAN, AND R.RABIN. 1957. Proteinase biosynthesis byStreptococcus liquefaciens. II. Purine, pyrimi-dine, and vitamin requirements. Can. J. Micro-biol. 3:553-558.

HODGMAN, C. D. 1960. Handbook of chemistry andphysics, 41st ed. Chemical Rubber PublishingCo., Cleveland.

LIu, T., N. P. NEUMANN, S. D. ELLIOTT, S.MOORE, AND W. H. STEIN. 1963. Chemicalproperties of streptococcal proteinase and itszymogen. J. Biol. Chem. 238:251-256.

RABIN, R., AND L. N. ZIMMERMAN. 1956. Proteinasebiosynthesis by Streptococcus liquefaciens. I.The effect of carbon and nitrogen sources, pH,and inhibitors. Can. J. Microbiol. 2:747-756.

SHEDLOVSKY, T., AND S. D. ELLIOTT. 1951. Anelectrophoretic study of a streptococcal pro-teinase and its precursor. J. Exptl. Med. 94:363-372.

VALLEE, B. L. 1960. Metal and enzyme interac-tions: correlation of composition, function,and structure, p. 225-276. In P. D. Boyer, H.Lardy, and K. Myrback [ed.], The enzymes,2nd ed., vol. 3. Academic Press, Inc., NewYork.

VALLEE, B. L., J. A. RUPLEY, T. L. COOMBS, ANDH. NEURATH. 1960. The role of zinc in car-boxypeptidase. J. Biol. Chem. 235:64-69.

WILLIAMS, R. J. P. 1959. Coordination, chelation,and catalysis, p. 391-442. In P. D. Boyer, H.Lardy, and K. Myrback [ed.], The enzymes,2nd ed., vol. 1. Academic Press, Inc., NewYork.

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