JOURNAL OF BACTERIOLOGY, Nov. 1971, p. 809-816Copyright i 1971 American Society for Microbiology

Vol. 108, No. 2Printed in U.S.A.

Amino Acid- -Naphthylamide Hydrolysis byPseudomonas aeruginosa Arylamidase

P. S. RILEY AND FRANCIS J. BEHAL

Department of Microbiology, Medical College of Georgia, A ugusta, Georgia 30902

Received for publication 21 July 1971

The intracellular and constitutive arylamidase from Pseudomonas aeruginosawas purified 528-fold by salt fractionation, ion-exchange chromatography, gel fil-tration, and adsorption chromatography. This enzyme hydrolyzed basic and neu-tral N-terminal amino acid residues from amino-,B-naphthylamides, dipeptide-,B-naphthylamides, and a variety of polypeptides. Only those substrates having an L-amino acid with an unsubstituted a-amino group as the N-terminal residue were

susceptible to enzymatic hydrolysis. The molecular weight was estimated to be71,000 daltons. The lowest Km values were associated with substrates having neu-

tral or basic amino acid residues with large side chains with no substitution or

branching on the ,B carbon atom.

The utilization of amino acid-f3-naphthyl-amides by bacteria has been a subject of investi-gation in our laboratories and those of other in-vestigators. These amino acid derivatives arehydrolyzed by arylamidases (or naphthyl-amidases), peptidase-like enzymes, to yield anamino acid and ,B-naphthylamine (#NA). In arecent paper (21), we reported that an arylami-dase from Pseudomonas aeruginosa was intracel-lular and constitutive, whereas the transportmechanism for amino acid-#NA was derepres-sible. P. aeruginosa cells grown on a minimalmedium produced the enzyme intracellularly butlacked the ability to take up exogenously sup-plied amino acid-,BNA. Brief incubation of thesecells with certain amino acids converted the cellsto ones having the ability to take up the sub-strate. During this incubation period, proteinsynthesis essential for amino acid-#NA transportoccurred. The transport process was energy de-pendent, and the amino acid moiety of the aminoacid-#NA was incorporated into bacterial pro-tein.

Other reports from our laboratory have beenconcerned with the purification and properties ofarylamidases from the gram-negative coccus,Neisseria catarrhalis. Behal and Cox reportedthat an unsubstituted amino group on the N-terminal L-a-amino residue was required for sus-ceptibility to enzymatic hydrolysis (5). Cox andBehal subsequently found that arylamidase hy-drolyzed dipeptide-,BNA and certain polypeptidesin a stepwise manner, beginning with the N-ter-minal residue, whereas those substrates having apenultimate residue of the D-configuration were

not hydrolyzed (11, 12). Arylamidases were ini-tially considered to be characteristic of gram-negative bacteria, but subsequent studies haveshown them to be present in gram-positive bac-teria as well (6, 20).

Aubert and Millet reported that L-leucyl-flNAhydrolase in Bacillus megaterium is more re-pressed during growth than during sporulation(2). Westly et al. have proposed the utilization ofsubstrate specificity patterns as a classificationtool for microorganisms (24). Similarly, Muftichas suggested a similar proposition for aid in dif-ferentiating mycobacteria (20). Burton et al.found that pathogenic and apathogenic strains ofleptospirae could be differentiated since signifi-cantly higher levels of naphthylamidase activitywere found in the pathogenic strains (10). Thearylamidase activity in Escherichia coli has beenlocated in the soluble fraction after cellular dis-ruption (23).

This report is concerned with our further in-vestigation into amino acid-ANA metabolism inP. aeruginosa and describes the purification andsome properties of the constitutive arylamidaseof this bacterium.

MATERIALS AND METHODS

Organism. P. aeruginosa cells were grown and har-vested as previously described (21).

Substrates. Amino acid-,BNA, dipeptide-,BNA, andpeptides were secured from Mann Research Laborato-ries, Inc., New York, N.Y., and from InternationalChemical and Nuclear Co., Burbank, Calif. The spe-cific rotations of the substrates used were in agreementwith literature values. All other chemicals used were ofthe highest commercial grade available.

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Protein determination. Protein was determined bythe method of Lowry et al. (17). Protein concentrationof individual fractions from column chromatographywas estimated by measuring the absorbance at a wave-

length of 280 nm.

Polyacrylamide gel electrophoresis. The alternatemethod of Davis for layering samples in 40% sucrosewas utilized (13). The buffer pH was 8.9, and the finalacrylamide concentration was 7%. Protein was detectedby staining with amido black in 7% acetic acid. En-zyme activity was detected by incubating the electro-phoresed gel at 37 C with a solution of 3 mM L-arginyl-f3NA containing 0.2 mg of Fast Blue B per ml. A redband appeared at the site of I3NA liberation. The gelswere stored in 7% acetic acid.

Paper chromatography. Chromatography was per-formed at 27 C for 15 hr on Whatman no. 3MM filterpaper. The ascending technique was utilized with a sol-vent system composed of n-butanol-glacial acetic acid-water (4:1:5, v/v). Free flNA was observed byviewing the chromatograms under ultraviolet light. Theamino acids and dipeptides were detected by sprayingthe chromatograms with a solution of 0.25% ninhydrinin acetone and subsequently heating the chromato-grams at 100 C for 10 min.

Molecular weight estimation. The molecular weightof the enzyme was estimated by a modification of thegel filtration method of Andrews (1). A PharmaciaK25/45 column, equipped with upward-flow adapters,was packed with Sephadex G-200 gel equilibrated with0.05 M potassium phosphate buffer, pH 7.0, containing0.1 mole of sodium chloride and 0.1 mole of boric acidper liter. The elution rate of the column was stabilizedby a metering pump, and each fraction contained 2 mlof eluant. The elution volume of Blue Dextran, approx-imately 80 ml, was used as the void volume of thecolumn. Several pure proteins of known molecularweight were employed as references for preparing a

standard curve.

Arylamidase assay. The rate of ,BNA release fromamino acid-,BNA was determined fluorometrically.Although unreacted substrate does not fluoresce appre-ciably, free ,NA does fluoresce intensely at 410 nm

when excited at 335 nm. The 4.0-ml reaction mixturecontained 40 gmoles of tris(hydroxymethyl)amino-methane-maleate buffer, 4.0 qmoles of amino acid-3NA (or dipeptide-,BNA), and enzyme. The pH of the

reaction mixture was varied as necessary. The rate ofarylamidase-catalyzed liberation of ,BNA at 37 C was

determined directly with a Beckman 77204 Ratio Flu-orometer and a strip chart recorder. A unit of arylami-dase activity is defined as that amount of enzyme thathydrolyzes 1.0 jimole of substrate per min under theseconditions. In some cases, a previously described color-imetric method (3) was employed instead.

RESULTS

Enzyme purification. A 100-g (wet weight)batch of cells was washed in 0.005 M potassiumphosphate buffer, pH 6.8, and then suspended in100 ml of the same buffer. The cells were thenruptured by ultrasonic treatment (Branson S125Sonifier) at 4 C. The resulting cell-free extractwas clarified by centrifugation at 40,000 x g.

Solid ammonium sulfate was added slowly withstirring to the clarified extraction until 30% ofthe saturation (168 g/liter) was reached. Afterstanding for 8 hr at 4 C, the precipitate was sedi-mented and the supematant fluid was brought to70% saturation (254 g/liter). After centrifuga-tion, the precipitate was resuspended in a smallvolume of 0.005 M potassium phosphate buffer,pH 8.6, and was dialyzed against 20 volumes ofthe same buffer for 12 hr. The dialyzing bufferwas changed each 3 hr. L-Arginyl-3NA was usedas the substrate to detect enzymatically activefractions throughout the purification procedure.The dialyzed fraction, containing 1.8 g of pro-

tein, was applied to a reverse-flow column (2.5by 100 cm; Pharmacia K25/100) packed withSephadex G-200 gel equilibrated at 4 C with a0.005 M potassium phosphate-0.10 M sodiumchloride buffer at pH 8.6. The flow rate was 0.3to 0.5 ml/min. Enzymatically active fractionswere pooled and concentrated by direct-pressuredialysis and dialyzed against 0.005 M potassiumphosphate buffer, pH 8.6, to prepare the samplefor ion-exchange chromatography.The pooled Sephadex column fractions, con-

taining 200 mg of protein, were applied to a di-ethylaminoethyl cellulose ion-exchange column(2.5 by 50 cm) which had been previously equili-brated with 0.005 M potassium phosphate buffer,pH 8.6. The column was eluted with 1,200 ml ofa potassium phosphate-sodium chloride gradientsystem. Initial sodium chloride molarity, potas-sium phosphate molarity, and pH were 0.0, 0.005,and 8.6, respectively: the respective limitingvalues were 0.4, 0.01, and 8.6. The column had aflow rate of I ml/min. Enzyme fractions werepooled, concentrated by pressure dialysis to avolume of 5 ml, and dialyzed against 0.001 Mpotassium phosphate buffer, pH 7.0.The sample was then applied to a column (0.5

by 40 cm) of Ca,(PO)2 (Hypatite C, ClarksonChemical Co., Inc., Williamsport, Pa.) whichhad been equilibrated with 0.001 M potassiumphosphate buffer. The sample was eluted with apotassium phosphate buffer gradient system; ini-tial and limiting phosphate molarities were 0.005and 0.05, respectively. The enzymatically activefractions were pooled, concentrated, and subse-quently applied to a second hypatite column andeluted with a stepwise gradient consisting of thefollowing molarities of potassium phosphate:0.005, 0.01, 0.015, 0.02, 0.025, and 0.03. The pHwas 7.0 throughout this procedure.The data for the purification procedure are

shown in Table 1. An overall 528-fold purifica-tion with a yield of 12% was obtained. The chro-matographic elution profiles from gel filtration,ion-exchange, and hydroxylapatite chromatog-raphy are illustrated (Fig. 1).

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TABLE 1. Purification of arylamidase

Step Total units Protein (mg) Specific activitya Purification

Cell-free extract 8.5 7,712 1.1 x l0-3 1Ammonium sulfate ppt (30-70% saturation) 7.9 1,968 4.0 x 10-3 3.5Gel filtration 7.1 228 31 x 10-3 28DEAE5 cellulose chromatography 4.6 58 80 x 10-3 73First hydroxylapatite chromatography 2.4 5 479 x 10-3 435Second hydroxylapatite chromatography 0.99 1.7 581 x 10-3 528

a Specific activity is expressed as units per milligram of protein.Diethylaminoethyl.

Acrylamide gel electrophoresis of the materialfrom the final step in the purification schemeproduced a single enzymatically active band. Theresults of gel electrophoresis of the purified en-zyme are shown on Fig. 2.Molecular weight estimation. Several proteins

of known molecular weight (human gammaglobulin, 160,000; bovine serum albumin, 67,000;ovalbumin, 45,000; bovine chymotrypsinogen,25,000; sperm whale myoglobin, 17,800; and cy-tochrome c, 12,400) were applied to and elutedfrom a Sephadex G-200 gel column as previouslydescribed above (Fig. 3).When P. aeruginosa arylamidase was chroma-

tographed, an elution volume to void volumeratio of 1.86 was obtained. This corresponded toa molecular weight of 7 1,000 daltons.

Substrate specificity. The relative rates of hy-drolysis of some ANA derivatives by arylamidasewere determined (Table 2) under conditions pre-viously established in studies in P. aeruginosa(21) and N. catarrhalis (5, 1 1). Certain basic andneutral amino acid-B3-naphthylamines were hydro-lyzed most readily, with L-arginyl-#NA and L-alanyl-#NA being most rapidly hydrolyzed. Therequirement for a primary amino group in the aposition was indicated by the fact that -y-glu-tamyl-,BNA and ,B-alanyl-,BNA were totally re-sistant to arylamidase-catalyzed hydrolysis. Sub-stitution of the a-amino group (e.g., N-acetyl-L-alanyl-,BNA) also rendered the substrates totallyresistant to hydrolysis. Furthermore, the lengthand branching of the carbon chain (e.g., L-leucyl-3NA versus L-isoleucyl-, L-valyl-, or glycyl-,BNA) greatly altered the susceptibility to enzy-matic hydrolysis.Hydrolysis of peptide-flNA and peptides. The

enzyme catalyzed release of #NA from the fouroptical isomers of alanyl-alanyl-,3NA was deter-mined by removal of samples of the reactionmixtures at intervals during the course of theincubation period. These samples were chroma-tographed to identify the reaction products. Thehydrolytic products of L-alanyl-L-alanyl-f3NAdetected were alanine, #NA, and some unreactedsubstrate. Analysis of the reaction products of

the sample containing L-alanyl-D-alanyl-f3NAshowed that, even after 17 hr of incubation, onlyalanine and alanyl-pNA were present. The iso-mers, D-alanyl-D-alanyl-fNA and D-alanyl-L-alanyl-,NA, were not susceptible to hydrolysiseven though incubated for 18 hr with enzyme(Fig. 4).

Catalytic activity upon short peptides was in-vestigated, as we thought that they were moreclosely related to the natural substrate of thisenzyme (Fig. 5). Complete hydrolysis of L-alanyl-L-alanyl-L-alanine to its component resi-dues occurred. L-Alanyl-L-alanyl-D-alanine wascleaved to form alanine and alanyl-alanine.However, L-alanyl-D-alanyl-L-alanine was com-pletely resistant to hydrolysis. Therefore, totalhydrolysis occurred only for those tripeptidescomposed entirely of L-amino acid residues.A comparison of the rate of ,NA release from

the substrates, L-alanyl-fBNA and L-alanyl-L-alanyl-#NA, was made (Fig. 6). The rate of ,BNArelease from L-alanyl-#NA was linear. However,with L-alanyl-L-alanyl-3NA, there was a lagperiod prior to reaching a linear release of ,BNA.Samples of these reaction mixtures, takenat various time intervals, were chromatographedto study the reaction products. In either sample,the only products detected were alanine and,BNA. Even a sample taken after 6 min of incu-bation from the L-alanyl-L-alanyl-fNA reactionmixture showed no trace of the dipeptide, alanyl-alanine.

Effect of substrate concentration on reactionvelocity. The effect of substrate concentration onthe rate of arylamidase-catalyzed hydrolysis wasdetermined for the amino acid-,BNA listed inTable 3. L-Alanyl-f3NA had the highest Vmaxvalue which slightly exceeded that of L-arginyl-,BNA. The lower Vmax values were obtained withthose substrates with ,B-branched R groups orwhen an R group was absent. The smaller Kmvalues were associated with the substrates con-taining amino acid residues with basic or non-polar R groups.Enzyme properties. The pH optimum for the

enzyme was 7.9, which is somewhat higher than

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1.0

0

0.8 8

-0.6 Z

m

-0.4m

04

.0.2

FIG. 2. Polyacrylamide gel electrophoresis of puri-fied arylamidase at pH 8.9. Gel on the right is stainedfor protein; gel on the left is stained for enzyme ac-

tivity. The lower band on the right gel is due to thetracker dye. The proteins migrated from the top to-ward the bottom of the gel.

-o.0 5

-0.

0.005

5S 100 150 200 25O

ELUTION VOLUME lul)

FIG. 1. Column chromatographic elution profiles.Top, gel filtration; middle, ion-exchange chromatog-raphy; and bottom, hydroxylapatite chromatography.Protein (A); enzyme activity (-). The column frac-tions were tested for erfzyme activity with the substrate,L-arginyl-fNA. The enzyme profiles were obtained byusing the colorimetric assay (3).

the values observed for arylamidases from N.catarrhalis (5, 11) and Sarcina lutea (4). Incuba-tion of the enzyme at pH values from 5.5 to 9.5did not irreversibly inactivate the enzyme, sinceadjustment to pH 7.9, after preincubation at theother values, resulted in activity levels similar tothat of the enzyme preincubated and assayed atthe pH optimum (Fig. 7).

I-

cr:

-J-

-i

v/v.

FIG. 3. Gel filtration molecular weight determina-tion. Standard reference materials were blue dextran(0); gamma globulin (-); bovine serum albumin (A);ovalbumin (U); chymotrypsinogen (O); myoglobin(A); cytochrome c (0); arylamidase (ON).

812 J. BACTERIOL.

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AMINO ACID-,-NAPHTHYLAMIDE HYDROLYSIS

TABLE 2. Hydrolytic activity ofarylamidase onvarious #NA derivativesa

Relative rateAmino acid-,BNA o yrlssof hydrolySiSb

L-Arginine ............ ............. 100L-Alanine ............. ............. 91L-Lysine ........................... 3 1L-MethiOnine ....................... 24L-LeuCine .......................... 22L-a-GlUtamate ...................... 7L-IsOleuCine ........................ 2L-Phenylalanine ..................... IL-Histidine ......................... IL-Glycine ............. ............. I

a The quantity of enzyme used in the assays was ad-justed to liberate 1 nmole of fNA per ml per min fromthe substrate, L-arginyl-3NA. Conditions of the fluoro-metric assay are presented in Materials and Methods.bThe arbitrary value, 100, was assigned to the rate

obtained with L-arginyl-fNA, the substrate most rap-idly hydrolyzed. The following amino acid derivativesof #NA were not susceptible to hydrolysis: L-a-aspar-tate, L-y-glutamate, L-proline, L-serine, ,B-alanine, L-threonine, L-valine, N-acetyl-alanine, D-alanyl-L-ala-nine, D-alanyl-L-alanine, and D-alanine.

The effect of several divalent cations upon therate of enzymatic hydrolysis of L-arginyl-/3NAwas investigated by comparison of duplicatesamples of the enzyme, one dialyzed in 0.05 Mpotassium phosphate buffer (pH 7.9) and theother in 0.1 M ethylenediaminetetraacetate (ED-TA), pH 7.9, followed by dialysis in the phos-phate buffer. Comparison of the activity of thesesamples with undialyzed control samples showedthat EDTA treatment caused a loss of approxi-mately 60% of the activity. Moreover, afterEDTA treatment, arylamidase was stimulated bymost of the metal ions tested (Table 4). Max-imum stimulation was provided by Mn2+ andMg2+. The enzymatic activity of the buffer-dia-lyzed sample (not treated with EDTA) was notstimulated by any of the metal ions. A stronginhibitory effect following the addition of Cu2+was noted in both samples. Increasing the con-centration of the metals up to I mm resulted in aloss of activity similar to that observed in theuntreated enzyme sample.

DISCUSSIONThere have been few reports on microbial aryl-

amidases, and the role(s) of these enzymes inbacterial physiology has not been established.There are, however, many reports on animal andhuman arylamidases for which physiologicalroles have been suggested (14, 16, 18, 19). Fel-genhauer and Glenner (15) proposed that the ratkidney enzyme functions as an angiotensinase or

an aminopolypeptidase and that the hydrolysis ofamino acid-,BNA is fortuitous. Hopsu-Havu andSarino (16) suggested that the aryl moiety ofthese substrates is not a significant factor in theformation of enzyme-substrate complexes. How-ever, Behal and Cox (5) observed that the rate ofenzymatic action upon amino acid-p-nitroanilideswas much lower than that for correspondingpNA derivitives, thereby indicating that the arylgroup in the substrate is also of importance.An understanding of the in vivo function of

arylamidase in microorganisms is an objective ofthe investigations in our laboratory. Our pre-vious report on this enzyme in P. aeruginosasuggested a function other than involvement inprotein synthesis for cellular multiplication (21).To study further the hydrolysis and metabolismof amino acid-,BNA in P. aeruginosa, an investi-gation of some of the characteristics of arylami-dase was indicated.

Polyacrylamide gel electrophoresis of the final

Rf

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FIG. 4. Composite chromatogram showing theproducts of arylamidase-catalyzed hydrolysis of theoptical isomers of L-alanyl-f3NA and L-alanyl- L-alanyl-j3NA. The assay mixture contained: substrate, 2Amoles; enzyme (amount required to completely hydro-lyze 2 ,umoles of L-alanyl-L-alanyl-f3NA in I hr), 0.1ml; and 0.05 M phosphate buffer (pH 7.9) to a finalvolume of 1.5 ml. The assay mixtures were inactivatedby boiling at various time intervals during the incuba-tion period.

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.Y .2 .2 2z .2 'C~~~~~~~~.2.PPO? O O P ? ? ? ?- 0 co~

Rf

0.30

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FIG. 5. Composite chromatogram showing theproducts of arylamidase-catalyzed hydrolysis ofalaninepeptides. The assay mixtures contained: substrate, 2Amoles; enzyme (amount required to completely hydro-lyze 2 Aimoles of L-alanyl- L-alanine in I hr), 0.1 ml;and 0.05 M phosphate buffer (pH 7.9) to a final volumeof 1.5 ml. The assay mixtures were then incubated at37 C. Samples from each assay mixture were inacti-vated by boiling at various time intervals during theincubation period.

E6.

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Time (min)

FIG. 6. Hydrolysis of L-alanyl-j3NA and L-alanyl-L-alanyl-j#NA by arylamidase. Each reaction mixturecontained 1.5 umoles of substrate. The enzyme concen-tration was adjusted to liberate I tAmole of #NA per

min from L-alanyl-#NA. The substrates were: L-alanyl-#NA (0); L-alanyl- L-alanyl-#NA (A). The lib-eration of j3NA at pH 7.9 was determined fluorometri-cally.

TABLE 3. Effect ofsubstrate concentration on reactionvelocitya

VmaxSubstrate K. (M) (moles x I1

x min- )

L-AlanylI-fNA .... 11.7 x 10-4 22.2 x 10-5L-Arginyl-#NA ... 5.0 x 10-4 15.4 X 10-5

L-Lysyl-fBNA ..... 5.3 x 10-4 7.1 x 10-5L-LeuCyl-fBNA .... 5.3 x 10-4 6.9 x 10-5L-Isoleucyl-#NA .. 6.7 x 10-4 1.9 X 10-5Glycyl-,BNA ...... 20.6 x 10-4 0.54x 10-5

aThe quantity of enzyme used in the assays was ad-justed to liberate I nmole of ,NA per ml per min fromthe substrate, L-arginyl-ftNA. Conditions of the fluoro-metric assay are presented in Materials and Methods.

5 0

45450

c 30 /

:iJ< 1S 1z

6 7 8 9

p H

FIG. 7. Effect of pH on arylamidase activity. En-zyme activity was determined at the indicated pH onassay mixtures containing I mM L-arginyl-#NA. Theincubation period was for 30 min at 37 C. The colon-metric assay (3) was used.

preparation from the purification procedure indi-cated the presence of one enzymatically activeprotein band. An estimated molecular weight ofapproximately 71,000 daltons was determinedwhich is nearly one-half of the value reported forthe enzymes from N. catarrhalis (5, 11). How-ever, it is quite close to the estimates made forarylamidases from S. lutea (4) and ox brain (9).The substrate specificity study (Table 2) indi-

cated that the N-terminal amino acid residue ofthe substrate must have an a-amino group. Thisamino group must also be of the L-configurationand unsubstituted. Those ,3NA substrates havingstraight-chain or y-branched R groups weremore susceptible to hydrolytic attack than theones with ,-branched or acidic R groups on theamino acid residue.

Although the highest Vmn,0 value was observedwith the substrate L-alanyl-#NA, the lower Km

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values were associated with substrates containinglarge R groups on the amino acid residue. Thus,the size of the R groups is an important factor inenzyme-substrate complex formation. That the Rgroup does not interact solely by hydrophobicbonding was indicated by the similar Km valuesof L-leucyl- and L-arginyl-3NA. The lower Vmaxvalues observed with L-isoleucyl- and glycyl-3NAshowed that ,B-branching or the absence of an Rgroup altered the substrate susceptibility to aryl-amidase hydrolysis.

In studies conducted on bacterial arylami-dases, there has been no evidence presented for a

metal ion dependency for activity; this is in di-rect contrast to studies on arylamidases derivedfrom animal tissues (7-9, 22). In the investiga-tion of N. catarrhalis arylamidase (11), Cox andBehal observed some stimulation of enzyme ac-

tivity after EDTA treatment. After treatment ofthe P. aeruginosa enzyme with EDTA, 60% ofthe enzyme activity was lost. However, after di-valent cation replacement, we observed a markedincrease in activity which exceeded that recordedwith the untreated enzyme. This stimulatory ef-fect has also been observed with bovine brainarylamidase (19). The effect did not appear to bemetallo-specific, as each of the metal ions testedproduced the stimulation in activity, with theonly exception being cupric ion. These resultsmay indicate that the native metal ions are veryfirmly bound, and only after EDTA treatmentdoes replacement with a more effective metal ionoccur. The effect produced by the metal may beone of inducing a conformational change ratherthan the direct participation in the reaction atthe catalytic site of the enzyme.The activity of this napthylamidase on the var-

ious optical isomers of alanyl-alanyl-fNA andalanine peptides demonstrated the absolute re-

quirement for the L-configuration in the N-ter-minal residue. Single sequential hydrolysis ofamino acid residues from the substrates was indi-cated by the following. (i) Hydrolysis of L-

alanyl-D-alanyl-/3NA yielded the reaction prod-ucts alanine and alanyl-JNA; (ii) hydrolysis ofL-alanyl-L-alanyl-D-alanine yielded the reactionproducts alanine and alanyl-alanine; and (iii) L-

alanyl-p3NA was rapidly hydrolyzed to yieldANA; whereas, in the hydrolysis of L-alanyl-L-alanyl-fNA, a lag period prior to the appearanceof ANA was noted and there was no release ofthe intact dipeptide, alanyl-alanine.

Although L-alanyl-L-alanyl-L-alanine was hy-drolyzed, L-alanyl-D-alanyl-L-alanine was nothydrolyzed; this may be the result of steric hin-drance of the ionized carboxyl group of thelatter. That the hindrance may be an ionic effectrather than simply spatial is indicated by the factthat the N-terminal residue of L-alanyl-D-alanyl-

TABLE 4. Effect of divalent cations on enzyme activitya

Reagent.NAEnzyme (R0agen ) liberated(l-) (units)

Controlb ..50Dialyzedc ..50Dialyzedc .................... FeCl2 20Dialyzedc .................... CoCl2 18Dialyzedc .................... MgCl2 28Dialyzedc .................... MnCl2 30Dialyzedc .................... CuCl2 6EDTA dialyzed . 20EDTA dialyzedd ........ ...... FeCl2 100EDTA dialyzedd ........ ...... CoCl2 137EDTA dialyzedd ........ ...... MgCl2 198EDTA dialyzed ........ ...... MnCl2 205EDTA dialyzedd .............. CuCI, 7

a The quantity of enzyme used in the assays was ad-justed to liberate I nmole of ,BNA per ml per min fromthe substrate, L-arginyl-fNA. Ten tliters of metal ionsolution was preincubated with 90 Aliters of enzymesolution for 30 min at 37 C. The control sample waspreincubated with 10 Aliters of water for 30 min at 37 C.After preincubation, these mixtures were assayed fluo-rometrically for activity as described in Materials andMethods.

b The original untreated enzyme preparation.c Enzyme dialyzed against 0.05 M phosphate buffer

(pH 7.9) for 12 hr.d Enzyme dialyzed against 0.1 M EDTA solution, pH

7.9, for 6 hr and then dialyzed against 0.05 M phos-phate buffer (pH 7.9) for 6 hr.

fNA is hydrolyzed, even though the penultimateresidue is of the D configuration.Thus, this enzyme does not cleave dipeptide-3NA to yield the products dipeptide and ,BNA,as has been reported for arylamidase of the pitui-tary (14). Therefore, there is a similarity inmechanism of action between this enzyme andthe arylamidases of N. catarrhalis (5, 11) andhuman liver (8).

LITERATURE CITED

l. Andrews, P. 1963. Estimation of the molecular weights ofproteins by Sephadex gel-filtration. Biochem. J. 91:222-233.

2. Aubert, J., and J. Millet. 1965. Etude d'une L-leucyl-3-naphthylamide hydrolase en relation avec la sporulationchez Bacillus megaterium. C. R. H. Acad. Sci. 261:4274--4277.

3. Behal, F. J., B. Asserson, F. Dawson, and J. Hardman.1965. A study of human tissue aminopeptidase compo-nents. Arch. Biochem. Biophys. 111:335-344.

4. Behal, F. J., and R. T. Carter. 1971. Naphthylamidases ofSarcina lutea. Can. J. Microbiol. 17:39-45.

5. Behal, F. J., and S. T. Cox. 1968. Arylamidase of Neis-seria catarrhalis. J. Bacteriol. 96:1240-1248.

6. Behal, F. J., and J. D. Folds. 1967. A comparative study ofbacterial alanine aminohydrolases. Biochim. Biophys.Res. Commun. 27:344-349.

7. Behal, F. J., and G. H. Little. 1968. Arylamidase ofhuman duodenum. Clin. Chim. Acta 21:347-355.

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8. Behal, F. J., G. H. Little, and R. A. Klein. 1969. Arylami-dase of human liver. Biochim. Biophys. Acta 178:118-127.

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