JOURNAL OF BACTERIOLOGY, Mar. 1975, p. 933-941Copyright 0 1975 American Society for Microbiology

Vol. 121, No. 3Printed in U.S.A.

Purification and Properties of a Serine Protease fromPseudomonas maltophilia

ROBERT S. BOETHLINGDepartment of Bacteriology, University of California, Los Angeles, California 90024

Received for publication 18 December 1974

The extracellular protease of Pseudomonas maltophilia was partially purifiedby ammonium sulfate precipitation and chromatography on Sephadex G-75 andBio-rex 70. Gel electrophoresis revealed minor impurities. The enzyme exhibitedthe following properties: (i) molecular weight, 35,000; (ii) A I%O = 10.8; (iii) isoelec-tric point, 9.3; (iv) pH optimum, 10.0; (v) S2o.11 = 3.47. The enzyme was rapidlyinactivated by ethylenediaminetetracetate, but activity could be partially re-stored with divalent cations. Of those tested, Ca,2+ Sr2+, Ba2+, Co2+, Cu2+,Mg2+, and Zn2+ were all effective. Both phenylmethylsulfonylfluoride anddiisopropylfluorophosphate were powerful inhibitors of protease activity, butL-1-tosylamide-2-phenylethylchloromethyl ketone, iodoacetic acid, and iodo-acetamide were without effect. The enzyme hydrolyzed the esters N-acetyl-L-tyrosine ethyl ester and a-N-benzoyl-L-arginine ethyl ester (BAEE) with Kmvalues of 10.4 and 3.4 mM, respectively. The hydrolysis of BAEE was also in-hibited by phenylarsonic acids. The kinetics of inhibition by m-nitrophenylarso-nate were of the mixed type, and the K, was 1.8 mM. The data followed a theo-retical curve for a 1:1 enzyme-inhibitor complex with a dissociation constant of1.8 mM. Inhibition by m-nitrophenylarsonate was pH dependent and followed atheoretical curve for the titration of a protonated group with a pKa of 7.0.

Extracellular proteases of microbial origin arethought to be instrumental in the degradationof complex protein substrates to amino acidsand peptides in nature. For the most part, theseenzymes are small molecules that can be iso-lated with comparative ease and in good yield.As a result, many of these enzymes have beencrystallized and extensively characterized withrespect to physicochemical properties and sub-strate specificity. The best studied as a groupare the microbial serine proteases, and amongthese the best known are the subtilisins. Thesubtilisins are alkaline proteases of broad speci-ficity produced by different strains of Bacillussubtilis. The most striking feature of theseenzymes is the structure of the active site,which is similar to that found in the pancreaticserine proteases, a group of enzymes of inde-pendent evolutionary origin (21).There are few reports of extracellular pro-

teases from gram-negative bacteria. Several ofthese appear to be metalloproteases that resem-ble bovine carboxypeptidase in their require-ment for Zn2+ (11, 12, 13, 16, 17). The enzymesfrom the psychrophile Escherichia freundii (16)and from Serratia sp. (12) resemble the sub-tilisins in that all have alkaline pH optima withcasein as substrate. In other respects, however,they are not similar. Extracellular enzymes

from Pseudomonas aeruginosa have been stud-ied by Morihara et al. (14, 15). This organismapparently produces at least two distinct pro-teases, one on alkaline protease (14) and theother an elastase (15) which also has caseino-lytic activity. Both are inhibited by ethylene-diaminetetracetate (EDTA) but not by diiso-propylfluorophosphate (DFP), and areotherwise dissimilar to the subtilisins. In a re-cent publication, Kato et al. (7) reported thepurification of four different enzymes from amarine psychrophile, Pseudomonas sp. no.548. One of these was an alkaline protease thatwas most active on casein at pH 10. The en-zyme was inhibited by both DFP and chelatingagents.

In this communication, the partial purifica-tion and properties of an extracellular proteaseproduced by a gram-negative isolate are de-scribed. The enzyme is a serine protease that ina number of ways appears to be closely relatedto the subtilisins. This work is the first step inan investigation of the process of enzyme secre-tion in bacteria.

MATERIALS AND METHODSBacteria and growth conditions. The organism

was isolated from enrichment culture containing de-natured hemoglobin as the sole carbon source by J.

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Lascelles. The inoculum was material derived fromsewage. The organism was identified as a strain ofPseudomonas maltophilia (6, 22) and was maintainedon slopes of 1.5% nutrient agar. Cells were grown in asalt-succinate-yeast extract medium which containedthe following: 0.2% (wt/vol) yeast extract, 0.2%(NH4)2SO4, 50 mM disodium succinate, 8.8 mMNaH2PO4, 25 mM K2HPO4, 1 mM CaCl2, and 1 mMMgCl2. The CaC12 and MgCl2 were added together asa concentrated sterile solution after autoclaving. ThepH was 7.2 and required no adjustment.

P. maltophilia is a strict aerobe. The bacteria weregrown to stationary phase (about 12 h) at 30 C in15-liter volumes of medium aerated with a sparger.Inoculation was made with 300 ml of a 12-h culturegrown at 30 C with aeration. Growth was followedturbidimetrically at 540 nm; an absorbance of 1.0 at540 nm was equivalent to 0.21 mg of cell protein/ml.Enzyme assays. Proteolytic activity in crude cul-

ture filtrates was assayed spectrophotometrically bythe Azocoll procedure. Sample volumes contained 0.1ml of enzyme solution and distilled water to a total of2.0 ml. The reaction was initiated by the addition of1.0 ml of a suspension of Azocoll at 10 mg/ml in 0.1 Mtris(hydroxymethyl)-aminomethane (Tris) buffer atpH 7.4, and the tubes were placed in a reciprocalshaker bath at 37 C for 15 min. The reaction wasterminated by filtration of the suspension throughcotton-plugged Pasteur pipets, and the absorbancewas measured at 520 nm.

During the purification of the enzyme and subse-quent experiments proteolytic activity was deter-mined by modifications of the method of Kunitz (8).In the purification, each assay contained 10 gl ofenzyme solution, 0.1 ml of 0.15 M CaCl2, and 1.0 ml ofcasein (Hammarsten) at 10 mg/ml in 0.1 M Trisbuffer, pH 7.4, in a final volume of 3.0 ml. Enzyme,CaCl2 and water to make 2.0 ml were preincubated for5 min at 37 C, and the reaction was initiated byaddition of the substrate. After incubation for 15 min,the assay was terminated by plunging the tubes intoice and immediately adding 2.0 ml of 10% trichloro-acetic acid. After at least 1 h, the precipitated caseinwas removed by filtration with Whatman GF/C disksand the absorbance was measured at 280 nm. Theenzyme preparation obtained after the final purifica-tion step will hereafter be referred to as purifiedenzyme.

For the determination of enzyme activity as afunction of pH, assays contained 0.1 ml of 0.15 MCaCl2, 1.0 ml of casein at 10 mg/ml in 50 mM NaCladjusted to pH 9.0, and 1.6 ml of buffer in a finalvolume of 3.0 ml. Buffers were of varying pH but theconductance was held constant at 3,000 + 300 umhosas measured by a Yellow Springs Instrument model 31Conductivity Bridge (Yellow Springs Instrument Co.,Inc., Yellow Springs, Ohio). The assay tubes werepreincubated for 5 min, and the reaction was allowedto proceed for 15 min as before. The reaction wasinitiated by the addition of 10 ml of purified enzyme at0.5 mg/ml and was terminated with 2.0 ml of 15%trichloroacetic acid.

In other experiments in which purified enzyme wasused, the casein assay procedure was similar to that

described for the purification, except that the sub-strate was made up in 50 mM glycine buffer at pH10.0, and assays were initiated by addition of enzymerather than substrate. One unit of protease activity isdefined as the amount sufficient to produce anincrease in absorbance at 280 nm of 1.0 as measuredby the casein assay. In all of the above procedures, (i)blank values were obtained by assay without enzymeand were substracted from experimental values, (ii)the absorbance of the blank never exceeded 0.1, and(iii) the assay was linear to an absorbance of about0.7.

For the determination of esterase activity the ratesof hydrolysis of N-acetyl-L-tyrosine ethyl ester(ATEE) and a-N-benzoyl-L-arginine ethyl ester(BAEE) were measured with a Radiometer modelTTT lc pH-stat equipped with a thermostattedreaction vessel (Radiometer, Copenhagen, Denmark).Reaction volumes were 5 ml, and titrations wereperformed in the absence of buffers at 37 C with 0.02N NaOH as titrating agent. All assays contained 0.1M KCl and 5 mM CaCl2. Standardization of thetitrating agent was performed with KH(103)2 asprimary standard. The assay was linear over a rangeof 20 to 200 Ag of enzyme per assay with BAEE, and 2to 20 jg with ATEE as substrate. Activity is expressedas milliequivalents of OH- released/minute per milli-gram of protein.

Protein assays. Protein was determined by themethod of Lowry et al. (10) with crystalline bovineserum albumin as standard.

Polyacrylamide gel electrophoresis. Electropho-resis was conducted in native gels of 7.5% acrylamideaccording to the method of Laemmli (9). A Hoefermodel SE-500 slab gel apparatus was used (HoeferScientific Instruments, San Francisco, Calif.). Gels of1.5-mm thickness were run at pH 8.9 for 3 h with nostacking gel, at a constant current of 20 mA. Thegels were stained with 0.1% Coomassie brilliant bluein 50% methanol-10% trichloroacetic acid for 2 h at37 C, and destained in 10% methanol-10% acetic acid.

Isoelectric focusing. Electrofocusing was con-ducted in an LKB column of 110-ml capacity accord-ing to the recommendations of the manufacturer(LKB Produkter AB, Bromma, Sweden). A pH gradi-ent of 3.5 to 10 was established by the use of 1%(vol/vol) carrier ampholytes (LKB) covering thisrange. The column contained as the anticonvectantmedium a stepwise gradient of 0 to 50% (wt/vol)sucrose, to which was added CaCl2 to a final concen-tration of 5 mM. Purified enzyme (11.7 mg in 0.5 ml)was added to one of the middle fractions duringpreparation of the sucrose gradient. Enzyme wasfocused at 4 C for 4 h at a constant voltage of 300 V,followed by 36 h at 400 V. Fractions of 2 ml werecollected.

Ultraviolet spectra. The ultraviolet spectrum ofthe purified enzyme was obtained with a Cary model14R spectrophotometer (Applied Physics Corp., Mon-rovia, Calif.).

Molecular weight determination. The molecularweight of the purified enzyme was estimated by themethod of Andrews (1). A column of Sephadex G-100(1 by 100 cm) was equilibrated with 10 mM Tris-5

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mM CaCl2, pH 7.4, and run at 10.8 ml/h. Calibrationstandards included ribonuclease A, myoglobin, a-chymotrypsinogen, ovalbumin, and bovine serum al-bumin. Each standard was applied as a solution of 4mg in 0.5 ml of buffer. All procedures were carried outat 4 C.

Sedimentation analysis. Sedimentation velocityexperiments were performed at 20 C in a Beckmanmodel E analytical ultracentrifuge with an AN-Drotor and Schlieren optics. Ultracentrifugation wascarried out at 44,770 rpm at a protein concentration of4 mg/ml, and at 52,640 rpm at 1, 2, and 3 mg/ml. Thepurified protease was first dialyzed exhaustivelyagainst 10 mM Tris-5 mM CaCl2, pH 7.4; the buffermade no measurable contribution to viscosity ordensity in the centrifugation.

Reagents. Sodium cacodylate, DFP, phenylmeth-ylsulfonylfluoride (PMSF), L-1-tosylamide-2-phenyl-ethylchloromethyl ketone (TPCK), iodoacetic acid,iodoacetamide, ATEE, BAEE, bovine pancreaticribonuclease A, ovalbumin, and bovine serum al-bumin were obtained from Sigma. Iodoacetic acidwas twice recrystallized before use. Phenylarsonicacid, o-nitrophenylarsonic acid, m-nitrophenyl-arsonic acid, p-arsanilic acid, and primary standardKH(10,)2 were gifts from A. N. Glazer. Casein (Ham-marsten) was obtained from Nutritional Biochemi-cals, Azocoll from Calbiochem, ethylenediamine fromEastman, and methylamine from Matheson, Cole-man, and Bell. Bovine pancreatic a-chymotrypsino-gen and sperm whale myoglobin were purchased fromMann. Bio-rex 70 was a product of Bio-Rad. SephadexG-25, G-75, and G-100 were products of Pharmacia.All other chemicals were of reagent grade.

RESULTSPurification. Extracellular protease activity

was measured during the growth of the orga-nism in batch culture (Fig. 1). In the complexmedium in which the cells were grown, proteaseactivity was not detected until early stationaryphase. When protease activity had reached amaximum, the culture was immediately chilledin an ice bath, and the cells- were removed bycentrifugation at 10,000 x g for 45 min. Thecells were discarded and the supernatant fluidwas retained. Unless indicated otherwise, alloperations during the purification of the en-zyme were carried out at 4 C. Solid ammoniumsulfate (561 g/liter of supernatant fluid) wasthen added with stirring, and the solution wasallowed to sit undisturbed overnight. The re-sulting precipitate was collected by centrifuga-tion at 10,000 x g for 30 min. The precipitatewas dissolved in approximately 100 ml of 10mM Tris-5 mM CaCl2, pH 7.4, and dialyzedagainst 4 liters of the same buffer for 48 h withat least three changes of buffer. A small insolu-ble residue was removed by centrifugation. The

TIME (lHZ)

FIG. 1. Growth and production of extracellularprotease by P. maltophilia. The organism was grownin batch culture, and protease activity was deter-mined by the Azocoll procedure. Samples of clearsupernatant fluid were obtained at intervals by cen-trifugation at 12,000 x g for 10 min. Symbols: celldensity, 0; protease activity, 0.

dialysate was concentrated by pressure ultrafil-tration with an Amicon UM-2 filter.The concentrated material from the previous

step was applied to a column of Sephadex G-75(3 by 170 cm) equilibrated with Tris-CaCl2buffer as before. The column was operated at32.4 ml/h, and 5-ml fractions were collected.Two components were eluted after the voidvolume; both contained activity but the secondwas much smaller and was more heterogeneouswhen examined by polyacrylamide gel electro-phoresis. Fractions from the first componentwith activity were pooled and concentrated bypressure ultrafiltration, and desalted at roomtemperature with a column of Sephadex G-25 (2by 20 cm) equilibrated with 10 mM sodiumcacodylate-5 mM CaCl2, pH 6.0.The Sephadex-derived material was fraction-

ated by chromatography on a column (2 by 20cm) of Bio-rex 70, equilibrated with cacodylate-CaCl2 buffer as before. The column was oper-ated at 32.4 ml/h, and 3-ml fractions werecollected. When the column was washed withthe same buffer, a small amount of proteinwithout protease activity emerged and wasdiscarded. A linear gradient of 0 to 0.2 M NaClin a total of 600 ml was then applied, and theenzyme emerged as a single component ofactivity at about 0.14 M NaCl. The peakfractions were pooled and concentrated by pres-

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sure ultrafiltration, and desalted with SephadexG-25 as before. The enzyme could be stored at-70 C for at least 6 months with no loss ofactivity. A typical purification is summarized inTable 1.

Criteria of homogeneity. Molecular sievechromatography was performed in connectionwith the determination of' molecular weight bygel filtration and did not reveal the presence ofcontaminating protein. Sedimentation analysisalso revealed a single protein component. Poly-acrylamide gel electrophoresis did, however,reveal minor impurities immediately below themajor band in the stained gel (Fig. 2). It ispossible that the contaminating material is aproduct of autodigestion since the intensity ofstaining increased with time of storage at 4 C ofthe purified enzyme and with the number oftimes that the preparation was thawed andrefrozen. A nearly identical gel pattern has beenobserved for the autodigestion products of sub-tilisin Carlsberg (23).

Ultraviolet spectra. The ultraviolet spec-trum of the purified protease is shown in Fig. 3.The vaiue of A "'0 calculated from the figure is10.8. The ratio of the absorbance at 280 nm tothat at 260 nm is 2.05, indicating the absenceof a significant quantity of nucleic acid.

Sedimentation analysis. Sedimentation ve-locity runs were performed at protein concen-trations of 1, 2, 3, and 4 mg/ml, and the valuesfor s20,, obtained were plotted versus proteinconcentration. A value for s201, of 3.47 wascalculated by extrapolation to zero protein con-centration. A single symmetrical peak was ob-served in the Schlieren patterns at all proteinconcentrations.Molecular weight. Gel f'iltration analysis

gave a value of 35,000 for the molecular weightof the purified protease (Fig. 4).

Isoelectric point. The apparent isoelectricpoint of the enzyme was 9.3. Failure to includeCaCl2 during the electrotocusing procedure re-sulted in a total loss of activity and of

TABLE 1. Purification of protease

Step Total Protein Sp act YieldStep units (mg) (U/mg) (%)

1. Ammonium 17,080 448 38.1 100sulfatea

2. Sephadex G-75 13,200 313 42.2 773. Bio-rex 70 6,270 84 74.7 37

a The protein concentration and caseinolytic activ-ity in the crude culture fluid were not measuredbecause of the high background absorbance at 280nm.

+MARKERDYE

FIG. 2. Polyacrylamide gel analysis of purified pro-tease. The direction of migration is from top tobottom.

reproducibility in the pattern of absorbance at280 nm of the eluted material; this effect wasprevented by the addition of 5 mM CaCl2.

Effect of pH and ionic strength on proteaseactivity. Maximum protease activity was ob-served at pH 10.0 with casein as substrate (Fig.5). Despite the high pH optimum the enzymedemonstrated a broad range of activity; at pHvalues of 6 and 12 the enzyme retained nearly50% of its peak activity. Since the ionic strengthwas held constant, the actual concentrations ofbuffer species necessarily varied with pH. Theconductance of the casein assay mixture wasapproximately 4,500 ,mhos; concentrations ofNaCl as high as 0.25 M, which corresponded toa conductance of 20,000 gmhos, were shown tohave no effect on protease activity.Temperature stability. The effect of temper-

ature on the stability of the purified proteasewas determined with purified enzyme at 0.5mg/ml in 10 mM Tris-5 mM CaCl2, pH 7.4.

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0.4

0.0

0.4

0.3

0.2

0.1

240 240 230 300 320WAVELENOTH (n"-

FIG. 3. Ultraviolet spectrum of purified protease.The spectrum was obtained with purified enzyme at0.50 mg/ml in 10 mM cacodylate-5 mM CaCl2 buffer,pH 6.0.

0.6RNas. A

myoglobin

0.5

oi- chymotrypsinog*n0.4

0.3 PROTEASE0.3

voalbumin

0.2

SSA

0.1

20 4s0 s0 ao 10.0

MOLECULAR WEIGHT x 10-4

FIG. 4. Estimation of molecular weight by gel fil-tration with Sephadex G-100. K. is a measure of theelution volume and is equal to ve - v0/vt - v0, whereVe is the elution volume, v, is the bed volume, and v. isthe void volume as determined by blue dextran. Theestimated molecular weight is 35,000.

Samples of the enzyme solution were incubatedat various temperatures for 15 min and wereassayed by the casein procedure at pH 10.0. The

enzyme can withstand temperatures as high as60 C for 15 min with no loss of activity.

Effect of EDTA and reactivation by metalions. In the presence of EDTA the enzyme israpidly inactivated (Fig. 6). Although it ispossible to recover 100% of the initial activity ifthe assay is carried out immediately in thepresence of Ca2+, the extent to which Ca2+-stimulated reactivation is possible also declinesand reaches a minimum of approximately 17%at 60 min. The ability of other divalent cationsto reactivate EDTA-treated enzyme is demon-strated in Table 2. All of the tested cationsexcept Hg2+ produce significant reactivation; infact, Ca2 , Sr2 , Co2+, and Ba2+ are essentially

0.5

E4

064

z

0.3

402

D-

x0-

0.1

* 7 a 9 10 11 12

FIG. 5. Effect of pH on protease activity. Theassays were carried out at constant ionic strength as

described. The ionic strength corresponded to a NaCIconcentration of approximately 50 mM. Symbols:ethylenediamine, 0; Tris, 0; methylamine, 0.

O.4

zI. 0.3

z

rt

7 0

10 20 40 50 00 120 100

T1ZE 6in )

FIG. 6. Effect of EDTA on protease activity, andreactivation by Ca2+. Purified enzyme at 0.5 mg/mlwas incubated with 10 mM EDTA in 10 mM Trisbuffer, pH 7.4. At intervals samples of 10 ,l (5 ,g ofenzyme) were removed and assayed with or without 5mM CaCI2 by the casein procedure, at pH 10.0.Symbols: with 5 mM CaCI2, 0; without CaCI2, 0.

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equivalent in this respect. These results suggestthat metal ion is required only for stability anddoes not participate in catalysis. An alternativeexplanation is that the addition of metal ionsother than Ca2+ to the assay mix results in theformation of a ternary complex of enzyme,metal ion, and EDTA at the enzyme surface,permitting the release of Ca2+ from EDTA.

Effect of protease inhibitors. The effects ofinhibitors of protease activity are summarizedin Table 3. Both PMSF and DFP are powerfulinhibitors of protease activity, but PMSF inhib-its more rapidly, as complete inhibition is

TABLE 2. Restoration of protease activity by metalionSa

Ion Reactivation (%)b

Ca2 ............................ 100Sr2 ...................... ...... 106Ba2... 99Co2....................... 96Cu2+ ............................. 91Mg2........................... 84Zn2+ .......................... 79Mn2 .......................... 33Hg2...................... . 4None .......................... 8

a Purified enzyme at 0.5 mg/ml was incubated for 5min at 4 C with 10 mM EDTA in 10 mM Tris buffer,pH 7.4. Ten-microliter samples (5 ug of enzyme) werethen assayed in the presence of 5 mM metal ions atpH 10.0 as described.

b The values given are the average of two determi-nations, normalized to the value for Ca2+ (i.e., Ca2+ =100%).

observed within 30 min at only a 4 times molarexcess of inhibitor over enzyme. The chymo-trypsin inhibitor TPCK has no effect at 20 timesmolar excess. The sulfhydryl protease inhibitorsiodoacetic acid and iodoacetamide are alsowithout effect, under conditions that would beexpected to reveal any inhibitory activity.

Esterase activity. The initial rates of hydrol-ysis of the ester substrates ATEE and BAEEwere measured as a function of substrate con-centration in a pH-stat, at pH 8.0. The valuesfor the kinetic parameters Km and Vmax werecalculated from the Lineweaver-Burk plots.These are, for ATEE, 10.4 mM and 16.7 x 10-2mEq of OH- released/min per mg of protein,and for BAEE, 3.4 mM and 0.37 x 10- 2mEq/min per mg. It is evident that ATEE is amuch better substrate for the purified proteasethan BAEE. The kinetic data for ATEE mustbe interpreted with caution, however, since theassay volumes contained 10% (vol/vol) p-diox-ane. No inhibitory effects from peroxides wereobserved, but p-dioxane is known to be acompetitive inhibitor of subtilisin.

Effect of phenylarsonic acids on esteraseactivity. The pentavalent organic arsenicalsphenylarsonic acid and its derivatives are po-tent inhibitors of serine esterases (3). For thisreason, a study of the effect of various phenylar-sonates on esterase activity was undertakenwith BAEE as substrate. The kinetics of hydrol-ysis of BAEE in the presence and absence of 2mM phenylarsonates are depicted in Fig. 7.Phenylarsonic acid appeared to be the mostpowerful inhibitor, but only m-nitrophenylar-sonate attained an apparent zero order rate of

TABLE 3. Effect of inhibitors on protease activitya

Relative sp actb

Inhibitor 20 x molar excess of inhibitor 4 x molar excess of inhibitor inh3b5itM

30 min 60 min 30 min 60 min 240 min

DFP ......................... 0.10 0.03 0.62 0.44PMSF ........................ <0.01 <0.01 <0.01 <0.01TPCK ........................ 1.00 1.00lodoacetic acid 1.00Iodoacetamide 1.00None ........................ 1.00 1.00 1.00 1.00 1.00

aPurified enzyme at 0.5 mg/ml in 10 mM Tris-5 mM CaC12, pH 7.4, was incubated at room temperatureunder the indicated conditions of inhibitor concentration and length of incubation. Molar ratios of inhibitor toenzyme were calculated by assuming a molecular weight of 35,000 for the protease. The indicated concentrationsof iodoacetic acid and iodoacetamide corresponded to a 1,000 times molar excess. DFP, PMSF, and TPCK weredissolved in 1-propanol, and iodoacetic acid and iodoacetamide in water, and added to samples of the enzymesolution. Ten-microliter samples (5 Ag of enzyme) were assayed at pH 10.0 as described. Controls containingonly samples of 1-propanol were run, and no effect on protease activity was observed.

b Activity is expressed relative to the control incubated without inhibitor.

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E

s 10 is 20 25

TIME (Imin)

FIG. 7. Hydrolysis of BAEE with and withoutphenylarsonates. The inhibitor concentration was 2mM and the initial concentration of BAEE was 40mM in all cases. Titrations were carried out at pH 6.1with 100 ug of purified enzyme. -----, Extension toshow apparent zero order rate of hydrolysis in thepresence of m-nitrophenylarsonate.

substrate hydrolysis within the time of assay. Inthe present of phenylarsonate, o-nitrophenylar-sonate and p-arsanilate, the rate of hydrolysiscontinued to fall throughout the assay period.None of the phenylarsonates tested exerted itseffect within the time of mixing of enzyme andinhibitor. The order of effectiveness of theinhibitors was phenylarsonate > m-nitrophen-ylarsonate > o-nitrophenylarsonate > p-arsani-late.To study the nature of inhibition by the

phenylarsonates, the hydrolysis of BAEE withand without 2 mM m-nitrophenylarsonate wasmeasured at varying substrate concentration(Fig. 8). It is apparent that the kinetics are ofthe mixed type. The results imply that thesubstrate binding site and the initial bindingsite for the inhibitor are not identical. Thedissociation of the enzyme-inhibitor complex issufficiently slow at pH 6.1 to permit a determi-nation of the proportion of free enzyme in asample containing enzyme and inhibitor bymeasurement of the initial rate of esterolysis inthe absence of added inhibitor (3, 4). If the rateof hydrolysis ofBAEE is measured with enzymepreincubated with inhibitor at varying concen-tration, it is possible to calculate a value of K,for the inhibitor. An apparent K, of 1.8 mM form-nitrophenylarsonate was thus determined

(Fig. 9). The data fit closely a theoretical curvethat was constructed by assuming a dissociationconstant of 1.8 mM for a 1:1 enzyme-inhibitorcomplex.The effect of pH on the inhibition of esterase

activity by m-nitrophenylarsonate is shown inFig. 10. The rate of hydrolysis of BAEE wasmeasured as a function of pH in the presenceand absence of 5 mM inhibitor; sufficient timewas allowed for the assays to attain apparentzero order rates of hydrolysis. It is evident thatthe inhibitory activity is profoundly dependenton pH. A theoretical curve for the titration of asingle protonated group with a pKa of 7.0compares favorably with the experimental val-ues. The results suggest that the binding of theinhibitor at the active site requires the presenceof a protonated histidine residue.

DISCUSSIONThe serine proteases have in common specific

and stoichiometric inactivation by certain or-ganophosphorus compounds (18). Within thisgroup are two classes of proteases, the pan-creatic serine proteases and the subtilisins,which, despite the homology of their activesites, exhibit completely different primarystructures (21). The results presented in thispaper strongly suggest that, like subtilisin, theextracellular protease of P. maltophilia is aserine protease of broad specificity. This en-zyme is of interest not only in view of the factthat it is the first such enzyme from a gram-neg-ative organism to have been studied in somedetail, but also because it appears to haveproperties that suggest both similarity to anduniqueness from other serine proteases.

Stabilization by Ca2+ is a common character-

-so -2S 25 so 75 100

* M{-1)[SAKE]

FIG. 8. Double reciprocal plot of BAEE hydrolysiswith and without m-nitrophenylarsonate (2 mM).Titrations were carried out at pH 6.1 with 100 ,g ofpurified enzyme. Symbols: With 2 mM m-nitrophen-ylarsonate, 0; without m-nitrophenylarsonate, 0.

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s0

,0

00

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2 3 4 5 6 7

FIG. 9. Determination of the K, for m-nitrophenyl-arsonate. Purified enzyme at 10 mg/ml was preincu-bated for 1 h at 4 C with m-nitrophenylarsonate ofvarying concentration. At the end of this period theinitial rates of esterolysis were measured in theabsence of inhibitor, with BAEE (40 mM) as sub-strate and 100 .sg of enzyme per assay. The preincuba-tion and assay were carried out at pH 6.1. The dataare expressed as activity without inhibitor minusresidual activity, divided by activity without inhibi-tor, or % inhibition. The curve is theoretical, assum-

ing a dissociation constant of 1.8 mM for a 1:1enzyme-inhibitor complex.

istic of extracellular enzymes. Although thedata presented here do not establish the preciserole of metal ions in the preservation of enzymeactivity, the rapid inactivation of the enzymeby EDTA clearly underlines the importance ofdivalent cations. Pseudomonas protease is alsoinhibited by the serine protease inhibitors DFPand PMSF, but not by the chymotrypsin inhibi-tor TPCK or the sulfhydryl protease inhibitorsiodoacetic and iodoacetamide. Pseudomonasprotease and the subtilisins compare favorablywith respect to isoelectric point and pH op-

timum; both enzymes may be classified asalkaline proteases. The isoelectric point of pseu-domonas protease is somewhat higher than therange exhibited by the subtilisins, but the pHprofile with casein as substrate is nearly identi-cal (5, 19). The two enzymes also share similarvalues for A ",' (2). A significant departure fromsubtilisin is revealed in the molecular weightof the purified protease. The molecular weightof 35,000 may be compared to values in the

J. BACTERIOL.

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4.0 4.4 4.5 7.2 7.* 6.0 6.4 3.6

PH

FIG. 10. Dependence of inhibition by m-nitrophen-ylarsonate on pH. The titrations were carried out atthe indicated pH with and without 5 mM inhibitor.The substrate was BAEE (40 mM), and titrationswere initiated by the addition of 100 ,ug of purifiedenzyme. The data are expressed as % inhibition, as inFig. 9. The curve is theoretical, for the titration of asingle protonated group with a pKa of 7.0.

range of 20 to 30,000 for both the pancreaticserine proteases and the subtilisins (5, 19).The hydrolysis of the esters ATEE and BAEE

and the inhibition of BAEE hydrolysis by phen-ylarsonic acids provide much useful informa-tion for a comparison of pseudomonas proteaseand subtilisin. If a molecular weight of 35,000 isassumed for the purified enzyme, it is possibleto calculate turnover numbers at maximal ve-locity (Kcat) for ATEE and BAEE hydrolysis.These are, respectively, 98 per s and 2.2 per s.The values calculated for K., Vax, and Kcat arewithin an order of magnitude of those given forthe subtilisins (2, 20). It should be noted,however, that the presence of impurities in thefinal enzyme preparation precludes strict inter-pretation of Vmax values and related kineticdata. More significant are the ratios of thevalues of these parameters for the esters ATEEand BAEE; the results are similar to those ex-pected for the subtilisins, and contrast sharplywith the properties of trypsin and chymotryp-sin (20).

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P. MALTOPHILLA SERINE PROTEASE

The pattern of inhibition by the phenylar-sonic acids also indicates a closer relationshipwith the subtilisins than with chymotrypsin (3).Like subtilisin, pseudomonas protease reactsslowly with all of the tested phenylarsonates,and the inhibition kinetics are of the mixedtype. This implies that the initial binding sitefor the inhibitor and the substrate binding siteare not identical. In contrast, chymotrypsinexhibits instantaneous inhibition by certainphenylarsonates, such as phenylarsonic acid,and inhibition is strictly competitive. Chymo-trypsin is also more strongly inhibited by p-arsanilate than by phenylarsonate; withpseudomonas protease and subtilisin the orderis reversed. The evidence suggests that, withrespect to the structure of the inhibitor-bind-ing site, pseudomonas protease and subtilisinare more similar to one another than either isto chymotrypsin. Finally, study of the pH de-pendence of inhibition by m-nitrophenylarson-ate and the dissociation constant for the pseu-domonas protease-inhibitor complex suggestthat the enzyme-inhibitor complex is a 1:1 in-teraction in which a histidine residue is in-volved in binding of the inhibitor, and there-fore, probably, in catalysis. The same holdstrue for subtilisin.

ACKNOWLEDGMENTSThis investigation was supported by Public Health Service

grant AM 11148 from the National Institute of Arthritis,Metabolism, and Digestive Diseases to J. Lascelles, in whoselaboratory this work was accomplished. During this investiga-tion I was a National Institutes of Health predoctoral trainee,supported by Public Health Service grant GM 01297 from theNational Institute of General Medical Sciences.

I am indebted to A. N. Glazer for supervision of this workand for many helpful discussions. I also wish to thank JuneBaumer for the ultracentrifugation.

LITERATURE CITED1. Andrews, P. 1965. The gel filtration behavior of proteins

related to their molecular weights over a wide range.Biochem. J. 96:595-606.

2. Glazer, A. N. 1967. Esteratic reactions catalyzed bysubtilisins. J. Biol. Chem. 242:433-436.

3. Glazer, A. N. 1968. Inhibition of "serine" esterases byphenylarsonic acids. J. Biol. Chem. 242:3693-3701.

4. Glazer, A. N. 1968. The time-dependent specific interac-tion of 4-(4'-aminophenylazo) phenylarsonic acid withsubtilisin. Proc. Nat. Acad. Sci. U.S.A. 59:996-1002.

5. Hagihara, B. 1960. Bacterial and mold proteases, p.193-213. In P. D. Boyer, H. Lardy, and K. Myrback(ed.), The enzymes, vol. 4. Academic Press Inc., New

York.6. Hugh, R., and E. Ryschenkow. 1961. Pseudomonas

maltophilia, an alcaligenes-like species. J. Gen. Micro-biol. 26:123-132.

7. Kato, N., F. Nagasawa, S. Adachi, Y. Tani, and K.Ogata. 1972. Purification and properties of proteasesfrom a marine-psychrophilic bacterium. Agric. Biol.Chem. 36:1185-1192.

8. Kunitz, M. 1946. Crystalline soybean trypsin inhibitor.H. General properties. J. Gen. Physiol. 30:291-310.

9. Laemmli, U. K. 1970. Cleavage of structural proteinsduring assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

10. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

11. McCullough, J. L., B. A. Chabner, and J. R. Bertino.1971. Purification and properties of carboxypeptidaseG . J. Biol. Chem. 246:7207-7213.

12. Miyata, K., K. Maejima, K. Tomoda, and M. Isono.1970. Serratia protease. I. Purification and generalproperties of the enzyme. Agric. Biol. Chem.34:310-318.

13. Miyata, K., K. Tomoda, and M. Isono. 1971. Serratiaprotease. III. Characteristics of the enzyme as metallo-enzyme. Agric. Biol. Chem. 35:460-467.

14. Morihara, K. 1963. Pseudomonas aeruginosa proteinase.I. Purification and general .properties. Biochim. Bio-phys. Acta 73:113-124.

15. Morihara, K., H. Tsuzuki, T. Oka, H. Inoue, and M.Ebata. 1965. Pseudomonas aeruginosa elastase. Isola-tion, crystallization, and preliminary characterization.J. Biol. Chem. 240:3295-3304.

16. Nakajima, M., K. Mizusawa, and F. Yoshida. 1974.Purification and properties of an extracellular protein-ase of psychrophilic Escherichia freundii. Eur. J. Bio-chem. 44:87-96.

17. Prescott, J. M., S. K. Wilkes, F. W. Wagner, and K. J.Wilson. 1971. Aeromonas aminopeptidase. J. Biol.Chem. 246:1756-1764.

18. Sanger, F. 1963. Amino-acid sequences in the activecenters of certain enzymes. Proc. Chem. Soc. 76-83.

19. Smith, E. L., and F. S. Markland, Jr. 1971. Subtilisins:primary structure, chemical and physical properties, p.561-608. In P. D. Boyer (ed.), The enzymes, vol. 3.Academic Press Inc., New York.

20. Smith, E. L., F. S. Markland, and A. N. Glazer. 1970.Some structure-function relationships in the sub-tilisins, p. 160-172. In P. Desnuelle, H. Neurath, andM. Ottesen (ed.), Structure-function relationships ofproteolytic enzymes. Munksgaard, Copenhagen.

21. Smith, E. L., F. S. Markland, C. B. Kasper, R. J.DeLange, M. Landon, and W. H. Evans. 1966. Thecomplete amino acid sequence of two types of sub-tilisin, BPN' and Carlsberg. J. Biol. Chem.241:5974-5976.

22. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966.The aerobic pseudomonads: a taxonomic study. J. Gen.Microbiol. 43:159-271.

23. Verbruggen, R., E. Duruisseau, and A. Baeck. 1974. Onthe heterogeneity and autodigestion of subtilisin"Carlsberg" as studied by agarose electrophoresis andGrabar-Williams immunoelectrophoresis. Biochim.Biophys. Acta 365:108-114.

941VOL . 121, 1975

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