JOURNAL OF BACTERIOLOGY, Sept. 1973, p. 1003-1010Copyright 0 1973 American Society for Microbiology

Vol. 115, No. 3Printed in U.S.A.

Characterization of Invertase Activity fromCariogenic Streptococcus mutans

HOWARD K. KURAMITSUDepartment of Microbiology, Northwestern University Medical-Dental Schools, Chicago, Illinois 60611

Received for publication 6 June 1973

Invertase activity from Streptococcus mutans GS-5 has been partially purifiedand shown to possess j3-fructofuranosidase specificity. The enzyme has a broadpH optimum between pH 5.5 and 7.5 and exhibits maximal activity at 37 C.Fructose, but not the glucose analogue a-methyl-D-glucoside, acts as a competi-tive inhibitor of the enzyme. None of the common glycolytic intermediates or

adenine nucleotides had any significant effect on enzyme activity. A molecularweight of approximately 47,000 was estimated for the enzyme. The enzyme doesnot appear to be catabolically repressed by glucose nor inducible by sucrose.

Higher specific activities of the enzyme are observed in fructose or glucose-growncells compared to sucrose-grown cells. These results are discussed in terms of theregulation of invertase activity in vivo.

Human smooth surface dental caries appearsto be initiated by certain strains of Streptococ-cus mutans in the presence of dietary sucrose(7). These cariogenic microorganisms are capa-ble of elaborating two enzymes which catalyzethe formation of high molecular weight polysac-charides from sucrose. Dextransucrase cata-lyzes the formation of insoluble dextran mol-ecules which play an important role in mediatingthe attachment of S. mutans to smooth surfaces(10). These organisms also produce the enzymelevansucrase which catalyzes the formation ofthe polyfructose levan from sucrose (3). Re-cently, Gibbons has demonstrated that one ofthese cariogenic strains, GS-5, can hydrolyze su-crose by an intracellular invertase-like enzymein addition to forming glucose and fructose con-taining polysaccharides from sucrose (9). Thepresence of this enzyme would enable the orga-nism to metabolize both the fructose and glu-cose moieties of sucrose to lactic acid which isan important factor in tooth demineralization(20).Like most invertase-like enzymes from bacte-

ria, the enzyme from S. mutans has not beenextensively characterized. In contrast, there isabundant information on the properties of thisenzyme from yeast and other fungi (14). Twotypes of enzymes with invertase activity havebeen demonstrated in microorganisms (14).Some invertases with Wl-fructofuranosidasespecificity (EC 3.2.1.26) appear to recognize thefructose moiety of sucrose and catalyze thehydrolysis of the glycosidic linkage on the

fructose side of the glycosidic oxygen. In con-trast, other invertases are classified as a-glucosidases (EC 3.2.1.20) and catalyze thehydrolysis of the glycosidic bond between theglycosidic oxygen and the a-glucoside residue ofsucrose.

Since the metabolism of dietary sucrose bycariogenic S. mutans plays an important role inthe development of dental caries, it was ofinterest to investigate the regulation of sucrosemetabolism in this organism. This report dem-onstrates that the invertase activity of strainGS-5 is due to a ,8-fructofuranosidase and dataconcerning the catalytic properties of the en-zyme and its regulation are presented. On thebasis of these results, the potential regulation ofthe enzyme is discussed.

MATERIALS AND METHODSGrowth of the organism. Human cariogenic S.

mutans GS-5 was obtained from R. J. Gibbons(Harvard Univ. Dental School). The organism wasmaintained on both Mitis salivarius agar (Fisher)slants and in brain-heart-infusion (BBL) broth at 4 C.Monthly transfers on agar slants and biweekly trans-fers in brain-heart-infusion broth were routinelymade. For growth of cells for enzyme extraction, 0.10ml of the organism maintained in broth was inocu-lated into 10 ml of fresh brain-heart-infusion medium.After ovemight growth at 37 C, this inoculum wasadded to 1 liter of the 2% Trypticase (BBL)-salts-glucose medium of Gibbons (10) supplemented with0.3% yeast extract (Difco) (TYS medium). The cul-tures were incubated at 37 C for 18 h.

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was obtained from Pharmacia and Biogel A-15 fromBiorad Labs. Raffinose was a product of Pfanstiehland melezitose, trehalose, turanose, maltose, anda-methyl glucoside were obtained from Sigma Chemi-cal Co. Cytochrome c, ovalbumin, chymotrypsinogen,and yeast hexokinase were also obtained from SigmaChemical Co. All other chemicals and reagents wereobtained from readily available commercial sources.Enzyme assays. Invertase activity was measured

in a standard incubation mixture containing 0.10 Mpotassium phosphate buffer, pH 7.0, 0.23 M sucrose,enzyme, and water in 1.0 ml. After incubation for 30min at 37 C the reaction was stopped by heating at100 C for 5 min in a Temp-Blok heater (ScientificProducts). Appropriate samples were then assayed forglucose by means of the Glucostat (WorthingtonBiochem. Corp.) method or for total reducing sugarsby the Somogyi-Nelson procedure (2). Free fructoseproduced during hydrolysis was calculated as thedifference between the glucose and total reducingsugar formed. Product formation was linear in the30-min incubation period.Enzyme activity in crude extracts containing le-

vansucrase and dextransucrase activities was meas-ured by taking advantage of the marked differences inthe pH-activity curves of the sucrose hydrolyzingenzymes. The amount of glucose produced fromsucrose by the action of invertase at pH 5.0 is 31% ofthat produced at pH 7.0 (Fig. 1), whereas the glucoseformed at pH 5.0 is 89% of that formed at pH 7.0 bythe action of levansucrase (H. Kuramitsu, unpub-lished data). Therefore, the following simultaneousequation can be solved to calculate the activity ofinvertase in crude extracts: glucose (invertase) +glucose (levansucrase) = total glucose at pH 7.0; 0.31glucose (invertase) + 0.89 glucose (levansucrase) =

total glucose at pH 5.0. The values calculated fromthis equation correlated well with the invertase activi-

e-

0

.)

pH

FIG. 1. Effects of pH on inuertase activity. Stand-ard assay conditions for total reducing sugar were

utilized with the following buffers adjusted to theappropriate pH ualues: 0, 0.1 M potassium phos-phate-citrate buffer; 0, 0.10 M potassium phosphatebuffer. The enzyme (1.8 Atg of fraction II) was in-cubated at the indicated pH values. The activities are

calculated relative to that for potassium phosphatebuffer, pH 6.0, (0.0029 U) which was set at 100%.

ties estimated from the profiles of crude extractschromatographed on Biogel A-15 columns.

Hexokinase (EC 2.7.1.1) activity was measured aspreviously described (1). Inorganic phosphate uptakewas measured utilizing the method of Gomori (11).Glucose-1-phosphatase (EC 3.1.3.10) activity was as-sayed by measuring the amount of glucose formed in a1.0 ml reaction mixture containing 0.10 M potassiumphosphate buffer, pH 7.0, 1.0 mM glucose-i-phos-phate, enzyme, and water.

Protein was determined by the method of Lowry etal. (13) utilizing human albumin, fraction V, as theprotein standard.Enzyme units. One unit of invertase activity is

defined as the amount of enzyme required to hydro-lyze 1.0 MAmol of sucrose per min under standard assayconditions.

Calibrated Sephadex G-100 column chromatog-raphy. Estimation of the molecular weight of theinvertase from S. mutans was carried out on a cali-brated Sephadex G-100 column essentially as de-scribed by Andrews (1). A coliumn (2.5 by 40 cm) ofSephadex G-100 was equilibrated with 0.01 M tris-(hydroxymethyl)aminomethane (Tris) buffer, pH 7.4,and washed overnight with the same buffer. A 1.0-mlsample containing invertase (0.79 U of fraction III),cytochrome c (3 mg), chymotrypsinogen (3 mg), oval-bumin (3 mg), and yeast hexokinase (8 U) was ap-plied to the column and developed overnight at 4 Cwith the same buffer at a flow rate of 40 ml per h. Thefractions (3 ml) were collected and proteins were

assayed either spectrophotometrically at 230 nm or

enzymatically (hexokinase and invertase).Partial purification of invertase activity. Crude

extracts of S. mutans were prepared by suspendingcells in 4 volumes of 0.05 M potassium phosphatebuffer, pH 7.0. The suspension was disrupted in a

Branson sonifier, model S75, with two 1-min periodsof sonic treatment at 6 amps. The suspension was

kept cool in an ice bath during this procedure. Themixture was then centrifuged at 20,000 x g for 15 minat 4 C. The supernatant fluid served as the crudeextract (Table 1).

Invertase activity could be readily resolved fromboth levansucrase and dextransucrase activities afterchromatography on Biogel A-15 columns (9). Thecrude extract was applied to a column (2.5 by 30 cm)of this resin previously equilibrated in 0.01 M Trisbuffer, pH 7.4, 0.04% sodium azide. Development ofthe column was accomplished utilizing the same

buffer and the temperature was maintained at 4 C.

TABLE 1. Partial purification of invertase

Stage Volume Activity Protein Sp. act.

(ml) (U/ml) (mg/ml) (U/mg)

I Crude extract 13.5 0.97 2.2 0.44II Biogel A-15 32.0 0.33 0.21 1.6

fractionIII Concentrated 3.1 2.7 0.17 16.0

DEAE-Sephadexfraction

7S IV \

25

0~~~~~~

5 6 7 8 9 Ic

1004 KURAMITSU

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Fractions (5.0 ml) containing significant invertaseactivity were pooled and assayed (Table 1).The pooled fractions were then applied to a column

(2.5 by 30 cm) of DEAE-Sephadex A-S0 previouslyequilibrated with 0.01 M Tris buffer, pH 7.4, and 0.1M KCl. The enzyme was eluted from the columnutilizing a 600 ml linear gradient from 0.10 to 0.40 MKCO in 0.01 M Tris buffer, pH 7.4. The fractionscontaining significant activity were pooled, concen-trated through an Amicon UM-10 ultrafilter, andassayed (Table 1). The partially purified enzyme wasquite stable for several months when stored at 4 C.

RESULTSEffect of carbon source on invertase

activity. Since invertase can be considered thefirst enzyme involved in the catabolism ofsucrose, it was of interest to determine whethersucrose was capable of inducing invertase activ-ity. When S. mutans was grown in the presenceof increasing levels of sucrose it was found thatinvertase activity was inversely related to thesucrose concentration (Table 2). Thus, sucrosedoes not appear to induce invertase activity inthis organism but instead represses the forma-tion of this enzyme. However, this later conclu-sion might be complicated by several additionalfactors. S. mutans elaborates both extracellularlevansucrase and dextransucrase activitieswhich can metabolize sucrose to both freeglucose and fructose (10). Therefore, the appar-ent repressive effect of high sucrose concentra-tions might be more directly related to eitherthe glucose or fructose levels produced from theextracellular hydrolysis of added sucrose. Totest this possibility, S. mutans was grown in thepresence of various levels of either glucose orfructose (Table 2). The results indicate thatcells grown in the presence of either hexosecontain higher invertase activities than cellsgrown in the presence of comparable levels ofsucrose. Furthermore, a 10-fold increase in the

TABLE 2. Effect of carbon source on invertaseactivity"

Carbon source (%) Sp. act.(U of protein per mg)

Sucrose (0.1).0.40Sucrose (0.5).0.20Sucrose (1.0).0.09Glucose (0.1).0.57Glucose (1.0).0.38Fructose (0.1).0.78Fructose (1.0).0.86

a Cells were grown in 500 ml ofTYS medium for 18h in the presence of the indicated carbon source.Extracts were prepared and assayed as describedunder Materials and Methods.

concentration of either glucose or fructose doesnot cause a significant decrease in invertaseactivity, whereas a similar increase in sucroseconcentration results in a fourfold decrease inspecific activity. These results suggest that theapparent repressive effects of sucrose are notdue to the formation of either glucose or fructosein the medium.

Invertase activity as a function of growth.Since the specific activity of many bacterialenzymes is dependent on the growth stage of theorganisms, it was of interest to examine inver-tase activity as a function of cell growth. Whencells were harvested at different stages of atypical growth curve, it was observed thatenzyme activity did not vary significantly in thelogarithmic and stationary growth stages (Table3). The low specific activity observed during theearly lag phase was the result of utilizing aninoculum grown ovemight in the presence ofsucrose. Therefore, invertase activity appears tobe produced equally well during all stages ofgrowth in S. mutans.

Characterization of invertase as a ,B-fructofuranosidase. Three different classes ofenzymes have been demonstrated in microorga-nisms which catalyze the hydrolysis of sucroseto hexoses. Sucrose phosphorylase catalyzes thephosphorolysis of sucrose to produce glucose-1-phosphate and free fructose (5). As mentionedpreviously, classical invertases which hydrolyzesucrose to glucose and fructose may exhibiteither fl-fructofuranosidase or a-glucosidasespecificity. It was therefore of interest to deter-mine the specificity of hydrolysis exhibited bythe invertase of S. mutans.The partially purified enzyme from S.

mutans did not possess sucrose phosphorylaseactivity since both free glucose and fructosewere produced in essentially equal amounts

TABLE 3. Invertase activity as a function of growtha

Sp. act.

Cell stage Incubation Absorbance (U of pro-T (h) (Klett units) tein permg)

Lag phase 4 78 0.13Early log phase 7.5 115 0.37Late log phase 9.5 310 0.60Stationary 27 323 0.32

aFour 500-ml samples of TYS medium containingglucose (0.4%) were inoculated with identical 5.0-mlinocula of strain GS-5 grown overnight in the samemedia but containing sucrose (1.0%). Growth wastraced on a Klett colorimeter utilizing a no. 54 filter.At the indicated intervals, a single flask was har-vested, the washed cells were extracted and assayedas previously described.

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during hydrolysis. Furthermore, no detectableinorganic phosphate uptake could be measuredduring the hydrolysis of sucrose. Finally, noglucose- 1-phosphatase activity (which mighthydrolyze glucose-i-phosphate formed duringphosphorolysis to free glucose) could be de-tected in purified preparations.The distinction between ,B-fructofuranosidase

and a-glucosidase activities can be made on thebasis of comparing the relative rates of hydrol-ysis of a series of 3-fructosides and a-glucosides(14). In addition to sucrose, raffinose (6-a-D-galactopyranosyl - a - D - glucopyranosyl - [ -D-fructofuranoside) was tested as an unsubsti-tuted f-fructofuranoside. The trisaccharidemelezitose (3 - a - D - glucopyranosyl - [d- D -fructofuranosyl- a-D-glucopyranoside) was uti-lized as a substituted 1-fructofuranoside. Thefollowing a-glucosides were also tested: a-methyl-D-glucoside, maltose (4-a-D-glucopy-ranosyl-D-glucopyranoside), turanose (3- a-D-glucopyranosyl-D-fructofuranoside), and tre-halose (1-a-D-glucOpyranosyl-a- D-glucopyrano-side).The results demonstrate that significant hy-

drolysis occurred only with those saccharidescontaining an unsubstituted f-fructofuranoside(Table 4). Furthermore, the significant hydrol-ysis of raffinose compared to melezitose is acharacteristic of enzymes possessing f3-fruc-tofuranosidase specificity (14). The observedfivefold difference in reactivity for the hydrol-ysis of sucrose compared to raffinose is similarto that reported for yeast [-fructofuranosidases(8). Therefore, these results are compatible with

TABLE 4. Specificity of hydrolysis of S. mutansinvertasea

Substrates Relative act. (%)

A. Unsubstituted,B-fructofuranosidesSucrose 100Raffinose 20

B. Substituted 3-fructofuranosideMelezitose 2.1

C. a-GlucopyranosidesTrehalose 0Turanose 4.3Maltose 0a-Methyl-D-glucoside 1.5

aInvertase (1.7 Ag of fraction III) was incubatedwith the indicated substrates at a concentration of0.23 M and assayed as previously described. Thehydrolysis of the reducing sugars, maltose and turan-ose, was measured by determining glucose produc-tion. All other rates of hydrolysis were carried out bydetermining the formation of total reducing sugar. Allrates were calculated relative to that produced in thepresence of sucrose in both assays (0.016 U).

a f3-fructofuranosidase rather than an a-glucosi-dase specificity for the invertase of S. mutans.pH optimum. Invertase activity as a function

of pH was measured utilizing potassium phos-phate-citrate and potassium phosphate buffers(Fig. 1). The enzyme exhibited a relativelybroad pH optimum from pH 5.5 to 7.5 withactivity decreasing rapidly at more acidic andalkaline pH values.Temperature optimum. The enzyme exhibi-

ted significant activity over a relatively widetemperature range (Fig. 2). Maximal activitywas observed at approximately 37 C.Heat stability. Since significant enzyme ac-

tivity could be detected at temperatures higherthan 37 C (Fig. 2), the heat stability of theenzyme was measured. When enzyme was in-cubated alone at 50 C, approximately one halfof the initial activity was lost in less than 2 min.Essentially no activity was demonstrated after 5min of incubation at this temperature. Whenenzyme was incubated at 60 C, one half of theinitial activity was inactivated within 20 s andno activity could be detected after 90 s ofincubation. The significant activities observedduring standard assays at 50 and 60 C (Fig. 2)were shown to be the result of the protectiveeffects of both potassium phosphate buffer andthe substrate sucrose on enzyme activity. Forexample, the presence of sucrose and phosphatebuffer stabilized 93% of the original activitywhen heated at 50 C for 2 min.

Sucrose saturation kinetics. When the sub-strate saturation kinetics were determined uti-lizing partially purified invertase preparations,normal Michaelis-Menten kinetics (Kin) wereobserved (Fig. 4). No cooperative effects involv-ing multiple sucrose sites could be detected.The Km for sucrose was calculated as approxi-

75 .

50

2%

._

0

251

v20 30 40 50

Temperature (OC)60

FIG. 2. Effect of assay temperature on invertaseactivity. The enzyme (1.8 Mg of fraction II) was

assayed under standard assay conditions for totalreducing sugar at the indicated temperatures. Theactivity at 37 C (0.0028 U) was designated 100% andall other rates were calculated relative to this value.

7/\

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100

ji80 \I I460 -

w- 20 \

I1 2 3 4 5

Time (min.)FIG. 3. Heat stability of invertase. Enzyme (1.8 Mg

of fraction II) was incubated alone in separate tubesat either 50 C (0) or 60 C (a). At the designated timeperiods, the tubes were immediately cooled in an

ice-water bath. Activity was measured under stand-ard conditions for total reducing sugar and the resid-ual activities were calculated relative to the unheatedcontrol (0.0024 U).

30

20

I/v30 .0

I/S

0 0.2 0.4 0.6

Sucrose (M)

FIG. 4. Velocity-concentration plot for invertase.Enzyme (3.6 Ag of fraction II) was incubated understandard conditions and assayed for glucose produc-tion except that the incubations were terminated after15 min. Inset, double-reciprocal plot of the same data.Initial velocities are expressed as micrograms ofglucose formed in 15 min.

mately 0.14 M under standard assay conditions.Inhibition by products. To obtain additional

information on the specificity of invertase ac-

tion, the effects of the products of the reactionon sucrose hydrolysis were examined. Fructoseproduced significant inhibition of enzyme activ-ity when added at concentrations approximat-ing the substrate concentration (Fig. 5). Con-centrations of fructose equivalent to substrateproduced essentially 50% inhibition. The kinet-ics of inhibition (Fig. 6) indicate that fructose isacting as a competitive inhibitor of the enzyme

as would be predicted from the ,B-fruc-tofuranosidase specificity of the enzyme.

Glucose could not be tested directly as aninhibitor at concentrations approaching that ofthe substrate since high levels of this productwould make measurement of hydrolysis ratesextremely difficult in the assays utilized in thisstudy. Therefore, a closely related nonreducingstructural analogue of glucose, a-methyl-D-glucoside, was utilized as a potential inhibitor.The results (Fig. 5) indicate that the glucoseanalogue is not an inhibitor of sucrose hydrol-ysis over a wide concentration range. Thissuggests that glucose itself may not be as potentan inhibitor of sucrose hydrolysis as is fructose.These results are also compatible with a ,B-fruc-tofuranosidase rather than an a-glucosidasespecificity for the invertase of S. mutans (15).

Nonspecific inhibitors of invertaseactivity. The effects of a variety of nonspecificenzyme inhibitors were tested on invertasepreparations. The enzyme was very sensitive toinhibition by the sulfhydryl reagent P-chloromercuribenzoate (P-CMB) (Fig. 7). Con-centrations of approximately 0.1 MM produced50% inhibition of enzyme activity. In contrast,two other sulfhydryl reagents, iodoacetate andN-ethylmaleimide, produced negligible inhibi-tion in this concentration range. The inhibitoryeffects of P-CMB could be reversed by theaddition of mercaptoethanol.Enzyme activity was also sensitive to the

protein denaturants urea and sodium dodecylsulfate. The former at 0.6 M and the latter at0.65 mM produced 50% inhibition of activity.Ethylenediaminetetraacetic acid had little ef-

100

so ./~60c0

40 /

20

0~02 0.4 0.6

Inhibitor (M)

FIG. 5. Effects of fructose and a-methyl-D-gluco-side on invertase activity. Activity was measuredunder standard conditions for glucose production inthe presence of varying concentrations of fructose (0)or a-methyl-D-glucoside (0) utilizing 3.6 ,g of frac-tion lI. Corrections for the effects of fructose ora-methyl-D-glucoside on the glucose determinationswere made with the appropriate controls.

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15 -

5 40,..- --

/~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~0 4 8

I/S

FIG. 6. Double reciprocal plot of the fructose in-hibitory effects on invertase activity.Enzyme (1.7 Agof fraction III) was assayed under standard conditionsfor glucose production in the presence (0) and ab-sence (0) of 0.22 M fructose. Velocity is expressed interms of mg of glucose produced during a 30-min incu-bation period.

1004,

i 80

060

> 40

t 20

00.2 0.4 0.6 0.8 1.0

PCMB (AM)FIG. 7. Sensitivity of invertase activity to inhibition

by P-CMB. Enzyme (6.1 pg protein of fraction II)was incubated with the indicated levels of P-CMBand assayed under standard conditions for totalreducing sugar production. The indicated activitieswere calculated relative to the enzyme activity(0.0097 U) measured in the absence of inhibitor.

fect on invertase activity suggesting that metal-lic cations may not be essential for activity. Ofparticular interest for an enzyme found in acariogenic microorganism was the observationthat fluoride ion tested at concentrations up to5.0 mM had little effect on invertase activity.

Search for allosteric regulators. Since in-vertase is the first enzyme involved in thecatabolism of sucrose, it was of interest todetermine whether this enzyme might be underallosteric regulation by a normal intracellularmetabolite. A number of glycolytic intermedi-ates and nucleotides were examined as possibleregulators of invertase activity. No significanteffects on sucrose hydrolysis were demonstrablefor the following intermediates at concentra-

tions up to 5.0 mM: nicotinamide adeninedinucleotide (reduced form), nicotinamide ade-nine dinucleotide phosphate (reduced form),nicotinamide adenine dinucleotide, nicotina-mide adenine dinucleotide phosphate, adeno-sine 5'- monophosphate, adenosine 5' -diphos-phate, adenosine 5'-triphosphate, cyclic adeno-sine 5'-monophosphate, glucose-6-phosphate,fructose - 6 - phosphate, fructose - 1,6 - diphos-phate, phosphoenolpyruvate. lactate, andpyruvate. Therefore, none of the common in-dicators of the energy state of the cell (19) northe intermediates or end products of glycolysisproduced any detectable effect on invertaseactivity.

Molecular weight determination. The mo-lecular weight of the invertase from S. mutanswas estimated after chromatography on cali-brated Sephadex G-100 columns (Fig. 8). Thecalculated molecular weight of 47,000 4 4,700agrees well with the value of 50,000 reported forthe same enzyme after chromatography oncalibrated agarose columns (9).

DISCUSSION

The results of the present study indicate thatthe invertase activity of S. mutans is notelevated by the presence of sucrose in thegrowth medium. In fact, significantly higherspecific activities are observed in cells grownwith fructose or glucose as carbon sources thanin cells grown with sucrose. Furthermore, inver-tase activity decreases as the sucrose concentra-tion in which the cells are grown is increased. Itthus appears that the presence of sucrose does

,80

6

.2

' 2

80 100 120Elution Volume (ml)

140

FIG. 8. Molecular weight estimation of invertaseafter chromatography on calibrated Sephadex G-100columns. Chromatography of invertase and standardproteins was carried out as described under Materialsand Methods. The molecular weight of invertase was

estimated as previously described (1). The markerproteins are (i), yeast hexokinase; (ii), ovalbumin;(iii), chymotrypsinogen; (iv), cytochrome c. Thearrow indicates the elution volume of the invertase.

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not result in an increase of invertase specificactivities in this organism. These results furthersuggest that the apparent repress,ive effect ofsucrose on invertase activity is not the result ofthe formation of either extracellular glucose orfructose and subsequent repression. The ob-served effects of sucrose on enzyme activitymight be due to a direct repressive effect on thesynthesis of the enzyme or, alternativelv. mavresult as an indirect consequence of cellularclumping and adhesion to glass observed insucrose-grown cultures of this organism (10). Atest of this latter possibility might be madeutilizing mutants of S. mutans defective ininsoluble dextran formation and hence in cellu-lar adhesion. The isolation and characterizationof such mutants is currently in progress in thislaboratory.The apparent repressive effects of sucrose on

invertase activity have also been observed inBacillus subtilis (18). Similarly, it was reportedthat the ,B-fructofuranosidase activity in theyeast Saccharomyces fragilis was depressed inthe presence of increasing concentrations ofsucrose (4). However, a direct repressive effectof sucrose on enzyme synthesis could not bedemonstrated since extracellular sucrose wasalso hydrolyzed to glucose and fructose in thesesystems.The invertase of cariogenic S. mutans ap-

pears to possess f-fructofuranosidase specific-ity since only compounds containing an unsub-stituted W-fructofuranoside residue are hydro-lyzed. In contrast, saccharides containing onlyan a-glucoside residue are not significantlyhydrolyzed. The relative rates of hydrolysis ofthe compounds tested in this study are similarto those observed for the well-studied ,B-fruc-tofuranosidase of yeast (12). Furthermore, addi-tional evidence for the specificity of the inver-tase is provided by the observation that fructoseand not the glucose analogue, a-methyl gluco-side, is a potent inhibitor of invertase activity(15).A comparison of the properties of the inver-

tase of S. mutans with similar enzymes fromother sacchrolytic bacteria is difficult in view ofthe limited data available from other bacteria.However, the ,8-fructofuranosidase of yeast is anextremely well characterized enzyme (14). Theinvertase of S. mutans appears to be active inmore alkaline environments than the compara-ble enzyme from yeast. The latter enzymepossesses maximum activity between pH 4.0and 5.5, whereas the enzyme from S. mutans ismost active between pH 5.5 and 7.5. Like theenzyme from yeast, the invertase from S.

mutans has maximal activity near 37 C. The Kmvalues for both enzymes are relatively high withthe enzyme from S. mutans having a value of0.14 M, whereas that of the yeast is approxi-mately 0.016 M (12). Both enzymes can bestrongly inhibited by the product fructose.However, in contrast to the yeast enzyme (15),the invertase from S. mutans is not inhibited bya-methyl-glucoside. This may indicate struc-tural differences in part of the active sites of thetwo enzymes. The inhibition produced by fruc-tose is of a competitive nature indicating thatthis product competes with sucrose for thesubstrate site of the enzyme. However, sincefructose may also act as an acceptor of thef3-fructosyl residues transferred in reactions cat-alyzed by ,B-fructofuranosidases (6) this expla-nation for the inhibitory effect may be anover-simplification.The enzyme from S. mutans behaves simi-

larly to the yeast enzyme in that both are verysensitive to inhibition by mercury compoundsand are relatively insensitive to iodoacetate.The molecular weight of approximately 47,000for the S. mutans enzyme cannot be comparedto other comparable bacterial enzymes sincedata on these latter enzymes are lacking. Themolecular weight of the protein moiety of theyeast enzyme has been estimated at 135,000(16). This is a minimum estimate since car-bohydrate appears to be associated with puri-fied preparations of this, cell-wall associatedenzyme. In this regard, it will also be of interestto determine the cellular localization of theinvertase activity in S. mutans.The results of this study indicating that none

of the common regulatory modulators of energymetabolism or glycolysis (19) effect invertaseactivity is consistent with previous observationson the regulation of other catabolic enzymes(17). As with these other systems, the regulationof sucrose catabolism apparently affects en-zyme synthesis rather than enzyme activity.However, in contrast to other catabolic en-zymes, the invertase of S. mutans does notappear to be inducible by its substrate norcatabolically repressed by glucose. Neverthe-less, in order to accurately describe the regula-tory mechanisms controlling invertase forma-tion in S. mutans it will first be necessary toexclude the effects of insoluble dextran forma-tion on cellular metabolism as well as to deter-mine whether the organism can readily synthe-size sucrose in glucose or fructose supplementedmedia. Experiments directed toward answeringthese questions are currently in progress in thislaboratory.

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ACKNOWLEDGMENTS

I acknowledge the expert technical assistance of SakieNakamura in the course of this investigation.

This investigation was supported by Public Health Servicegrant DE-03258 from the National Institute of Dental Re-search.

ADDENDUM IN PROOF

Evidence for f3-fructofuranosidase activity in crudeextracts of S. mutans was also presented (McCabe,Smith, and Cowman, Arch. Oral Biol. 18:525-531,1973) after this manuscript was submitted for publi-cation.

LITERATURE CITED

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2. Ashwell, G. 1957. Colorimetric analysis of sugars. p.85-86. In S. P. Colowick and N. 0. Kaplan (ed.).Methods in enzymology, vol. 3. Academic Press Inc.,New York.

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