Biochem. J. (1975) 149, 187-197 Printed in Great Britaini

187

The Purification and Properties of Peroxidase in Mycobacterium tuberculosis H37Rv and Its Possible Role in the Mechanism of Action of Isonicotinic Acid Hydrazide By B. GAYATHRI DEVI,* M. S. SHAILA,f T. RAMAKRISHNAN and K. P. GOPINATHAN Microbiology and Cell Biology Laboratory, Indian Institute ofScience, Bangalore 560012, India (Received 15 November 1974) Peroxidase from Mycobacterium tuberculosis H37Rv was purified to homogeneity. The homogeneous protein exhibits catalase and Y (Youatt's)-enzyme activities in addition to peroxidase activity. Further confirmation that the three activities are due to a single enzyme was accomplished by other criteria, such as differential thermal inactivation, sensitivity to different inhibitors, and co-purification. The Y enzyme (peroxidase) was separated from NADase (NAD+ glycohydrolase) inhibitor by gel filtration on Sephadex G-200. The molecular weights of peroxidase and NADase inhibitor, as determined by gel filtration, are 240000 and 98000 respectively. The Y enzyme shows two Km values for both isoniazid (isonicotinic acid hydrazide) and NAD at low and high concentrations. Analysis of the data by Hill plots revealed that the enzyme has one binding site at lower substrate concentrations and more than one at higher substrate concentrations. The enzyme contains 6g-atoms of iron/mol. Highly purified preparations of peroxidases from different sources catalyse the Y-enzyme reaction, suggesting that the nature of the reaction may be a peroxidatic oxidation of isoniazid. Moreover, the Y-enzyme reaction is enhanced by 02. Isoniazidresistant mutants do not exhibit Y-enzyme, peroxidase or catalase activities, and do not take up isoniazid. The Y-enzyme reaction is therefore implicated in the uptake ofthe drug. One of the most important biochemical differences between isoniazid (isonicotinic acid hydrazide)sensitive and -resistant strains of mycobacteria is that resistance to isoniazid results in the loss of peroxidase (Tirunarayanan & Vischer, 1957; Hedgecock & Faucher, 1957; Steif et al., 1958) and catalase (Middlebrook, 1952, 1954, 1957; Middlebrook et al., 1954; Cohn et al., 1954) activities. However, the presence of catalase activity in a small but significant fraction of low-level-isoniazid-resistant mutants and occasionally in high-level-resistant mutants has also been reported (Winder, 1964). Since all of the isoniazid-resistant mutants analysed so far have lost their peroxidase activity, there is a better correlation of the sensitivity of the organism to isoniazid to its peroxidase activity than to its catalase activity. The agreement between isoniazid resistance and peroxidase activity exists in atypical mycobacteria also, in that all these atypical mycobac*

Present address: Department of Microbiology and

Immunology, University of Califormia, Los Angeles, Calif., U.S.A. t Present address: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Conn., U.S.A.

Vol. 149

teria that are naturally resistant to isoniazid are also peroxidase-negative. In some ofthese strains, catalase activity is present (Andrejew et al., 1956, 1959, 1960; Winder, 1960). Most of the drug-resistant strains analysed have been isolated after repeated or graded exposure to isoniazid, either clinically or in the laboratory, and it is not certain whether these mutations have occurred in a single step or in multiple steps. Earlier workers (Szybalski & Bryson, 1952; Cohn et al., 1954) reported that resistance to fairly high concentrations of isoniazid (20,ug/ml or more) has arisen by a single-step mutation. However, according to Tsukamura (1960), high-level resistance in Mycobacterium tuberculosis var. hominis requires two steps. Unless this fact (that mutation has arisen as a single step) is established, no meaningful correlation of the lost biochemical characteristics can be made. Six isoniazid-resistant mutants of M. tuberculosis H37Rv, which are presumably single.step mutants, have lost their peroxidase activity and their capacity to take up isoniazid (Sriprakash & Ramakrishnan, 1970); their catalase activity, however, has not been analysed. These isoniazid-resistant strains were classified into two categories, based on the

188 behaviour of a protein which acts as an inhibitor for the enzyme NADase (NAD+ glycohydrolase, EC 3.2.2.5) and which is capable of binding to isoniazid; in one class of mutants, the NADase inhibitor lost its sensitivity to isoniazid, whereas in the other class, the NADase inhibitor retained the parental phenotype, so that it can still bind isoniazid. These results also link the NADase inhibitor to peroxidase activity and the development ofisoniazid resistance, although their exact relationships are not known. Previously we have presented evidence (Gayathri Devi et al., 1972, 1974) of an enzyme system, referred to as Y enzyme* in M. tuberculosis H37Rv, similar to the one observed in Bacille Calmette-Guerin (Youatt & Tham, 1969a,b). The Y enzyme catalyses the formation of a colourless product by using isoniazid and NAD, which turns yellow on acidification and is strongly implicated in the mechanism of action of isoniazid. When the properties of this enzyme were analysed, the enzyme was found to be inhibited by the known inhibitors of peroxidase. Further, both Y-enzyme and peroxidase activities were lost in the two isoniazid-resistant mutants belonging to different categories. To investigate how these two activities are interrelated, purification of these enzyme activities was attempted. Their relationship to the enzyme catalase was also investigated. In this paper evidence is presented to show that catalase, peroxidase and Y-enzyme activities are catalysed by a single protein. The NADase inhibitor, though associated with these enzyme activities until the final step of purification, appears to be a different protein, distinct from the latter. Materials and Methods Materials NAD was from the V.P. Chest Institute, New Delhi, India. Sephadex G-25 and G-200 were from Pharmacia Fine Chemicals, Uppsala, Sweden; GSH, p-chloromercuribenzoate, ,B-mercaptoethanol, N-ethylmaleimide, bovine liver catalase (crystalline), horseradish peroxidase types II and VI, lactoperoxidase, DEAE-cellulose and Amido Black were from Sigma Chemical Co., St. Louis, Mo., U.S.A.; isoniazid and H202 were from Sarabhai Merck Chemicals, Baroda, India; o-dianisidine was from Fluka A.G., Buchs, Switzerland. Acrylamide was from Matheson Co., Norwood, Ohio, U.S.A. and NN'-methylenebisacrylamide was from American Cyanamid Co., Wayne, N.J., U.S.A. Other reagents for gel electrophoresis were from Eastman Kodak Ltd., Rochester, N.Y., U.S.A.; Coomassie Brilliant Blue was from Colab Laboratories Inc., Glenwood, 111., U.S.A. * Abbreviation: Y enzyme, Youatt's enzyme, which catalyses a reaction of isoniazid in the presence ofNAD.

B. GAYATHRI DEVI AND OTHERS

Methods Organism, culture conditions and preparation of cell-free extract. M. tuberculosis H37Rv (strain no. N.C.T.C. 7416) was grown as a surface culture at 37°C, and a cell-free extract was prepared as described previously (Gayathri Devi et al., 1974). Protein was determined by the method of Lowry et al. (1951). Enzyme assays. The Y enzyme was assayed as described previously (Gayathri Devi et al., 1974). The standard assay system contained, in a final volume of 1 .0ml: 50,pmol of Tris-HCl buffer, pH 8.0; 0.15,umol of NAD; 1.5pmol of isoniazid; and the enzyme. The reaction was carried out at 37°C for lh and was terminated by the addition of 0.2 ml of 60% (w/v) HC104. Precipitated proteins were removed and the yellow colour of the supernatant was measured at 420nm. One unit of Y enzyme is defined as that amount required to give an increase in absorbancy of 1.0 at 420nm, under the given assay conditions. Peroxidase activity was assayed by the method described in the Worthington Manual (Worthington Biochemical Corp., Freehold, N.J., U.S.A., 1966). The rate of decomposition of H202 with odianisidine as the hydrogen donor was determined spectrophotometrically by measuring the colour development at 460nm. One unit of peroxidase is defined as the amount of enzyme decomposing 1 umol of H202/min at 25°C. Catalase activity was assayed by following the disappearance of H202 spectrophotometrically at 240nm (Beers & Sizer, 1952). One unit of catalase activity is defined as the amount of enzyme decomposing 1 umol of H202/min at 25°C. Preparation and assay of NADase from M. tuberculosis H37Rv. The purification of NADase, up to the (NH4)2SO4 precipitation step, was carried out as described by Gopinathan et al. (1964). NADase activity was assayed by following the disappearance of NAD by using the cyanideaddition method. The enzyme assay system contained, in a final volume of 0.7ml, 100.umol of potassium phosphate buffer, pH 6.5, 0.25,umol of NAD and the enzyme. The reaction was carried out for 1 h at 37°C and was terminated by the addition of 3ml of 1 MKCN. The absorbance of the NAD-cyanide complex at 325 nm was measured spectrophotometrically. For controls, NAD was added at the end of incubation period just before the addition of cyanide. One unit of the enzyme is defined as the amount which will cleave 0.1 ,umol of NAD/min at 37°C. Assay of NADase inhibitor activity. This was assayed as described by Gopinathan et al. (1966). The reaction mixture, in a final volume of 0.7ml, contained: 100lumol of potassium phosphate buffer, pH6.7; NADase from M. tuberculosis, 1 .5-2units; 1975

PEROXIDASE AND ISONIAZID ACTION and the inhibitor, in amounts sufficient to give 30-60% inhibition of NADase. At the end of 20minm incubation at 28°C, 0.1 ml (0.25,umol) of NAD was added and the reaction mixture was incubated for an additional I h at 370C. The reaction was terminated bytheaddition of 3 ml of 1 M-KCNand the absorbance at 325nm was recorded. Appropriate controls of enzyme, inhibitor and substrate were included. A unit of the inhibitor activity is defined as that amount needed to cause 50 % inhibition of one unit of NADase. Enzyme purification. Unless otherwise stated, all operations were carried out at 0-4°C. (1) Desalting with Sephadex G-25. Samples (150ml) of cell-free extracts (13 OOOg supernatant) were added to lOg of Sephadex G-25. The gel was allowed to swell for 15-20min in the cold (5°C), centrifuged and the supernatant was collected. (2) DEAE-cellulose chromatography. Extracts (lOOml) treated with Sephadex G-25 were loaded on a DEAE-cellulose column (1.5cmrx 1OOcm), previously washed and equilibrated with 0.5mMpotassium phosphate buffer, pH7.0. The flow rate was 75ml/h. The adsorbed proteins were eluted from the column in a stepwise manner with potassium phosphate buffer, pH7.0, of increasing molarity (0.05-0.1 M). The buffers were changed only when the elution of all the 280nm-absorbing substances with that particular buffer was complete. The 0.1 Mbuffer eluate showed NADase-inhibitor activity, catalase, peroxidase and Y-enzyme activities. This fraction was designated as the DEAE-cellulose eluate. (3) Gel filtration. The DEAE-cellulose eluate was concentrated to 2-4ml by freeze-drying, and applied to the top of a column (1.2cmx48.5cm) of Sephadex G-200. The gel filtration was carried out with 0.02M-potassium phosphate buffer, pH7.0; 2.8ml fractions were collected. The individual fractions were monitored for theabsorbancy at 280nm and assayed for peroxidase, catalase, Y-enzyme and NADase-inhibitor activities. The fractions were pooled according to the enzymic activities and rechromatographed on another Sephadex G-200 column (2.2cmx440cm) under similar conditions, except that 4.4ml fractions were collected. Estimation of molecular weight. The approximate molecular weight of the enzyme was estimated by gel-filtration of the protein on a calibrated column of Sephadex G-200, as described by Andrews (1964). Determination of iron content. The iron content of the purified enzyme was determined by the bathophenanthroline method, after wet ashing, as described by Van de Bogart & Beinert (1967). Acrylamide-gel electrophoresis. Polyacrylamide-gel electrophoresis was carried out by using Tris (0.05 M)glycine (0.3M) buffer, pH18.3, or /5-alanine (0.035M)acetic acid (0.014M) buffer, pH4.3, on 7.5% acrylVol. 149

189

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B. GAYATHRI DEVI AND OTHERS

190 amide gels (Ornstein & Davis, 1962). The gels were stained with Amido Black or Coomassie Brilliant Blue and destained by diffusion by using 7% acetic acid. The gels were also stained for enzymic (peroxidase) activity (Shannon et al., 1965). Results Purification of the enzyme activities The results of purification of the enzyme activities are presented in Table 1, which shows that catalase, peroxidase and Y-enzyme activities were always associated with the same protein components. The gel-filtration patterns of these activities are presented in Fig. 1. Although Sephadex G-200 gel filtration gave three distinct protein peaks, all the three enzyme activities were associated with a single protein peak. At this stage of purification the NADaseinhibitor activity (V. 47.6-64.4ml) was well separated from the three former activities (Ve 28-42ml). The active fractions were separately pooled and rechromatographed on a Sephadex G-200 column. The chromatographic profiles obtained are shown

in Fig. 2 (for Y enzyme, catalase and peroxidase) and Fig. 3 (for the NADase inhibitor). A single coincident peak of protein and the enzyme activities was obtained; with NADase inhibitor, there may be a small amount of contaminating protein. No NADaseinhibitor activity could be detected in the catalaseperoxidase-Y-enzyme fraction or vice versa. Thus it is clear that the three latter activities are distinct from the NADase inhibitor. Properties of the purified Y enzyme Optimum pH. The relation between pH and the enzyme activity was studied, and the results are presented in Fig. 4. The enzyme showed a broad pH optimum ranging from 7.6 to 8.6. Influence oftemperature. The optimum temperature for enzymic activity was 38°C. Further increase in temperature resulted in the loss of activity, and at 55°C there is total inactivation of the enzyme. The energy of activation, calculated from the Arrhenius plot of the temperature-activity relationship was 11 340J/mol. Homogeneity. The purified enzyme, on rechromatography on a Sephadex G-200 column, gave a single

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Fraction no. Fig. 1. Sephadex G-200-/iltration profiles of catalase, peroxidase, Y-enzyme and NADase-inhibitor activities from M. tuberculosis H37Rv The 0.1 M-phosphate eluate from DEAE-cellulose was freeze-dried, dissolved in 1 .5 m1 of 0.02M-potassium phosphate buffer, pH7.0, and applied to a Sephadex G-200column (1.2cmx48.5cm). Thcflowratewas6ml/hand the fraction size was 2.8ml. Catalase, peroxidase, Y-enzyme and NADase-inhibitor activities were determined by using standard assay conditions.

1975

Plate 1

The Biochemical Journal, Vol. 149, No. 1 (b)

(a)

(c)

EXPLANATION OF PLATE I

Polyacrylamide-gel electrophoresis of M. tuberculosis H37Rv peroxidase- Y-enzyme-catalase The enzyme (1OO,ug) was submitted to electrophoresis on 7.5%Y acrylamide gel for 60min, (a) at pH8.3 (Tris-glycine buffer) and (b) at pH4.3 (,6-alanine-acetic acid buffer). (c) Enzyme staining (for peroxidase activity) after polyacrylamide-gel electrophoresis. The electrophoresis was carried out in Tris-glycine (pH8.3) buffer; 2-5,ug of protein was subjected to electrophoresis at 24°C under standard conditions for 60 min. After the run, the gels were immersed in 0.005 M-o-dianisidine in 0.01 M-phosphate buffer, pH 6.0. for 2min and in 0.005M-H202 for 10min. The zone of peroxidase activity appeared as a brownish-red band on the gel. B. GAYATHRI DEVI AND OTHERS

(Facing p. 190)

191

PEROXIDASE AND ISONIAZID ACTION

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Fraction no. Fig. 2. Rechromatography of Y-enzyme, catalase andperoxidase activities on Sephadex G-200 The active fractions 10-15 (in Fig. 1) were pooled and rechromatographed on a second Sephadex G-200 column (2.2cmx41.8cm); 4.4ml fractions were collected. Standard conditions were used for measuring the enzyme activities.

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Fraction no. 3. Fig. Rechromatography of NADase-inktbitor activity on Sephadex G-220 Fractions showing NADase-inhibitor activity (fractions 17-23 in Fig. 1) were pooled and rechromatographed on a second Sephadex G-200 column (2.2cmx41.8cm); 4.5ml fractions were collected and assayed for NADase-inhibitor

activity.

7

9

demonstrated on the gels by enzyme staining (Plate Ic). There was only a single activity band found on the gel, and no isoenzymes could be detected. Stability. The purified enzyme is stable for several

months when stored at -20°C.

coincident peak of protein and enzyme activity. The enzyme preparation was also electrophoretically homogeneous at both alkaline pH (8.6) and acid pH (4.3) when tested by polyacrylamide-gel electrophoresis (Plates la and lb). Peroxidase activity was Vol. 149

8

pH Fig. 4.pH-activity curveforpure Yenzyme at 370C Potassium phosphate buffer was used in the pH range 6-7 and Tris-HCl buffer was used for pH values 7-9.

Molecular weight. The approximate molecular weight of the enzyme was estimated by gel filtration on a column of Sephadex G-200. The molecular weight of the enzyme is 240000, and that of the NADase inhibitor 98000.

B. GAYATHRI DEVI AND OTHERS

192

(b) 0.8

0.7

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0.4k 0.3 [ 0.21

0.1 (

240

260

280

300

320

400

500

600

700

Wavelength (nm) Wavelength (nm) Fig. 5. AbsorptionspectrumofM. tuberculosisH37Rvperoxidase-Y enzyme-catalase (a) U.v. region: enzyme concentration is 0.0455% in potassium phosphate buffer, pH7.0. (b) Visible region: enzyme concentration is 0.18% in potassium phosphate buffer, pH7.0.

Absorption spectra. The purified enzyme was reddish-brown in colour. The u.v. and visible spectra of the purified enzyme are shown in Figs. 5(a) and 5(b). The [maximum absorption in the u.v. region was in the characteristic peak at 278nm; the visible absorption spectrum showed the characteristics of a haem protein, with a Soret absorption maximum at 408nm and with at least two more smaller peaks in theregion of490-500nm and 620-630nm. Effect of varying substrate concentrations. (1) Isoniazid concentration. The effect of varying isoniazid concentrations on the enzyme activity was studied and the results are presented in Fig. 6. The activity continued to increase in the wide range of concentrations used. A Lineweaver-Burk plot of the data revealed a biphasic response, as shown in Fig. 6, inset (a) in the lower concentration ranges, and inset (b) in the higher concentration ranges, of the substrate. At non-saturating concentrations of isoniazid, the Km obtained was 7.69 10-M and the corresponding Vma.. obtained was 2.86. At nearsaturating concentrations of isoniazid, the Km increased to 13.9x 10-5M and the Vn,a. to 3.93. x

A Hill plot of the substrate saturation data of the Y enzyme showed h 0.9 at lower substrate concentrations and 1.5 at higher substrate concentrations. These results indicate that at lower concentrations of substrate there is one binding site, which may increase to more than one at higher substrate concentrations. (2) Saturation of the enzyme with NAD. The NADsaturation curve of the enzyme resembled the substrate isoniazid pattern. A Lineweaver-Burk plot of the NAD-saturation data also showed a biphasic response (Fig. 7), giving Km values of 4.25 x 10-5M and 10.7 x 10-5M at lower concentrations and near-saturating concentrations respectively. The corresponding Vmax. values obtained were 3.08 and 4.35. A Hill plot of the NAD-saturation data also showed two slopes with h values of 0.8 and 1.8. Inhibition studies with picolinic acid hydrazide. Picolinic acid hydrazide, an analogue of isoniazid, is inhibitory to the Y enzyme. Its inhibitory effect (at different concentrations) on a wide range of isoniazid concentrations was studied, and the results were analysed graphically by double-reciprocal plots 1975 =

193

PEROXIDASE AND ISONIAZID ACTION

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10 x [Isoniazid](M) Fig. 6. Effect ofisoniazid concentration on Y-enzyme activity Standard assayconditions were used. The concentration ofNAD was 1 pmol (99.4mM). Incubation time was 45min, at 37°C. Lineweaver-Burk plot of the data, in the lower ranges of substrate concentrations [inset (a)] and higher ranges of substrate concentrations [inset (b)]. v is expressed as units of enzyme activity as defined in the Materials and Methods section.

inhibitory pattern observed could not be explained by any simple mechanisms. Requirement for oxygen. Since the enzyme reaction was stimulated by bubbling oxygen through the assay mixture, the requirement for oxygen was analysed in detail. The results are presented in Fig. 9, which

A 0.2 0.3 0.4 10-5/[NAD] (M-1)

0.5

0.6

Fig. 7. Lineweaver-Burk plot for NAD The velocity ofthe reaction was measured at various NAD concentrations by using the standard assay method, with 3,umol of isoniazid. Incubation time was 45min at 37°C. v is expressed as units of enzyme activity as defined in the Materials and Methods section.

(Fig. 8). At high concentrations of substrate (above 0.15mM) the inhibition was competitive, whereas at low concentration ranges (results not shown) the Vol. 149

shows clearly that the rate of reaction was higher in a 100% oxygen atmosphere, and was linear with time at least until 2h of incubation, whereas under anaerobic conditions, practically no reaction was observed. Effect of reducing agents. Since the Y enzyme catalysed an oxidative type of reaction, the effect of certain reducing agents such as GSH, I8-mercaptoethanol, dithiothreitol, L-cysteine, sodium dithionite and L-ascorbic acid on the enzyme reaction was studied. All the compounds tested, except ascorbic acid, inhibited the enzyme activity strongly (65-100 %) at 1 mM; dithionite caused 85% inhibition, even at 0.1

mM.

Effect of thiol-group reagents. The effect on the enzyme reaction of thiol inhibitors such as p-chloromercuribenzoate, N-ethylmaleimide and iodoacetate was studied. p-Chloromercuribenzoate and N-ethylmaleimide completely inhibited the enzyme at 1 nm concentration, whereas iodoacetate did not show any significant inhibition. Reversal of inhibition by GSH or mercaptoethanol could not be demonstrated,

194

B. GAYATHRI DEVI AND OTHERS

0.240.16

0.08

0

30

60

90

120

Time (min) Fig. 9. Oxygen requirement for Y-enzyme activity Standard assay conditions were used. The assays were carried out in Thunberg tubes under appropriate gaseous conditions. 0, Control; 0, oxygen; A, anaerobic.

0

12

6

18

10-3/[S] (M-1) Fig. 8. Effect ofpicolinic acid hydrazide Thestandardassaysystemcontainedtheindicatedamounts of picolinic acid hydrazide. The isoniazid concentrations were varied and the Lineweaver-Burk plots for isoniazid at different concentrations of picolinic acid hydrazide are given: (1) picolinic acid hydrazide, (2) 5lig, (3) 10g (4) 20pg, (5) 30pg, (6) 150pg. v is expressed as units of enzyme activity as defined in the Materials and Methods section. as these reagents were themselves inhibitory at low concentrations. Effect of various inhibitors. The effect of metalchelating agents on the enzyme was studied, and sigificant inhibition by reagents such as o-phenanthroline, aca'-bipyridyl, diethyl dithiocarbamate and 8-hydroxyquinoline was observed. Various other inhibitors, such as NaCN, hydroxylamine, Na2S, Na2SO3 and NaN3, also inhibited the

strongly (complete inhibition at 1mm and 65-85 %/* inhibition at 0.1 mM concentration of these inhibitors). These results suggest that metal ions such as iron or copper may be involved in the enzyme activity; however, these metal ions themselves

enzyme

did not show any stimulation. Metal content. Analysis for iron in the pure enzyme showed the presence of 6g-atoms of iron/mol of enzyme.

Comparison of properties of catalase, peroxidase and Y enzyme. Since all these activities were associ-

ated during the purification procedure, with parallel recoveries and a parallel constant rise in their specific activities (see Table 1), it appeared that the three activities are the properties of a single protein. Further, the final preparation was a single homogeneous protein (both electrophoretically and chromatographically). The ratios of the specific activities of the three activities at different stages of purification were constant throughout. The thermal-inactivation profiles of the three activities of the enzyme were studied and the results are presented in Figs. 10 and 11. It is evident that the inactivation of all the three functions follows the same pattern.

Y-enzyme reaction with pure preparation of horseradish peroxidase and lactoperoxidase Since the Y-enzyme, peroxidase and catalase activities were associated with a single protein, pure preparations of catalase (ox liver) and peroxidase (horseradish peroxidase as well as lactoperoxidase) fromvastly different sourcesweretested to seewhether they could catalyse the Y-enzyme reaction in the presence of isoniazid and NAD. It was observed that horseradish peroxidase (both type II and the more purified type VI) and lactoperoxidase catalysed the Y-enzyme reaction, whereas the catalase preparations did not show this activity. Comparison ofthe properties ofM. tuberculosis H37Rv peroxidase and horseradish peroxidase From the results presented above, it is clear that pure preparations of peroxidases from vastly different

1975

PEROXIDASE AND ISONIAZID ACTION

195 sources can catalyse the Y-enzyme reaction in th presence of isoniazid and NAD. Therefore it was decided to compare the properties (such as pH opti-

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20 0

S4N Ca

30

40

50

60

80

70

Temperature (°C) Fig. 10. Thermal stability of M. tuberculosis H37Rv peroxidase- Y enzyme-catalase The enzyme solution in 0.O5M-Tris-HCl buffer, pH 8.0, was incubated at various temperatures for 1 min. (The enzyme was preheated in a boiling-water bath to the required temperature and maintained for 1 min at the same temperature.) The residual activity was measured at 370C. A, Y-enzyme activity; 0, catalase activity; 0, peroxidase activity.

2

0

3

Time (min) Fig. 11. Stability ofM. tuberculosis H37Rvperoxidase-Yenzyme-catalase activity at 60QC The enzyme solution in 0.05M-Tris-HCl buffer, pH8.0, was mnaintained at 60'C for various periods and the residual enzymes activity was measured. A, Y-enzyme activity; *, catalase activity; 0, peroxidase activity.

Table 2. Comparison ofpropertiesofpurifledM. tuberculosisH37Rvperoxidase andhorseradishperoxidae (type VI)

Properties *I. 2. *3. 4. 5. 6.

M. tuberculosis H37Rv peroxidase 7.5-8.6 240000

pH optimum Molecular weight Temperature optimum

370C

Y-enzyme activity (units) Peroxidase activity (units) R2 value

2.9 44.45 1

Concn. ... 1 mM *7. Inhibition with: Cyanide Azide Hydroxylamine Suiite

Sulphide *8. Inhibition wi-th cheators:

awg'-fipyridyl

8-Hydroxyquinoline o-Phenanthroline Diethyl dithiocarbamate EDTA *9. Reducing agents: GSH Dithiothreitol

fi-Mercaptoehanol *10. Thiol-group reagents: p-Chkomercuribenzoate N-Efiyhnaleimide lodoacetate * With respect to Y-enzyme activity. Vol. 149

100.0

94,8 100,0 100.0 100.0

Horseradish peroxidase 7.5-8.6 40000 450C 1.52 840

0.lmm

1mM

0.1mm

88.4 34.6 65.4 71.6 65.5

83.0 47.0

44.1 21.0 86.2 54.0 54.7

79.3 85.8 87.3 96.1 67.3

35.5 51.5 79.9

30.6 65.8 76.6

15.1 16.9 15.6

100.0 100.0

24.6 83.9

0

0

26.6 57.0

100.0 80.0 74.7

80.1 89.4 82.0 100.0 73.2

30.4 55.3 63.2 40.0

30.0 61.0

17.1 19.2 23.0

82.0

94.9 90.8 0

60.1

24.7 72.0 0

196 mum and sensitivity to various inhibitors) of M. tuberculosis H37Rv peroxidase (Y enzyme) and horseradish peroxidase. The results are presented in Table 2. The two enzymes differed very much in their size, and temperature optimum for the catalytic activities; however, they show remarkable similarities in their pH requirements (a broad optimum range, pH7.5-8.6) and their sensitivity to various classes of inhibitors.

Discussion The catalase and peroxidase activities of mycobacteria have attracted the attention of several workers, particularly since these activities appeared to be involved in the mechanism of isoniazid action. Nevertheless, whether the two activities are due to one protein or to two different proteins has been the subject of some controversy. In Bacille CalmetteGuerin, these two activities are similar in their heat stability, pH optimum and sensitivity to various inhibitors (Winder, 1960). Moreover, the ratio of catalase to peroxidase remained almost constant during the partial purification steps. In contrast, Andrejew & Renard (1968) reported that catalase and peroxidase activities from Mycobacterium avium could be separated by electrophoresis on cellulose acetate strips. The data in the present paper clearly show that, in M. tuberculosis H37Rv, the two activities are catalysed by a single protein. Further, the Y-enzyme activity is also associated with the same protein. The thermal-inactivation profiles and the specific catalytic activities of the three functions are identical in the final, homogeneous protein preparation. Although the isoniazid-sensitive parental strain of M. tuberculosis H37Rv is positive for catalase, peroxidase and Y-enzyme activities, all the three activities are lost in the two classes of isoniazidresistant mutants. These mutants were classified on the basis of the NADase inhibitor (Sriprakash & Ramakrishnan, 1970). A role for the NADase inhibitor in isoniazid action has also been suggested previously (Bekierkunst & Bricker, 1967; Sriprakash & Ramakrishnan, 1970). It has been clearly demonstrated in the present paper that the NADase inhibitor can be completely separated from catalaseperoxidase-Y-enzyme activities, and that the two proteins differ significantly in size; whereas the mol.wt. of the latter enzyme is 240000, the mol.wt. of the NADase inhibitor is 98000, as estimated by gel-filtration on Sephadex G-200 columns. Anothercloselylinked property whichaccompanies isoniazid resistance is the loss of uptake of isoniazid. Wimpenny (1967a,b) has suggested that a 'hydroperoxidase' may be involved in the uptake of isoniazid. The uptake of isoniazid by the organism,

B. GAYATHRI DEVI AND OTHERS as well as the Y-enzyme reaction in Bacille CalmetteGu6rin, is greatly stimulated in the presence of 02 (Youatt & Tham, 1969a,b). The Y-enzyme activity of M. tuberculosis H37Rv is also stimulated by °2, and there is no reaction under anaerobic conditions. Further, both the Y-enzyme reaction and isoniazid uptake are inhibited by isoniazid analogues, and by inhibitors such as cyanide, azide and hydroxylamine. Therefore it is likely that the Y-enzyme reaction is involved in the uptake of isoniazid. The kinetic behaviour of the enzyme was.somewhat anomalous, and two Km values can be calculated for isoniazid, one in the lower and one in the higher concentration ranges. The two values differ by a factor of nearly two. A similar situation was observed when the Km values for NAD were determined. Analysis of the kinetic data by Hill plots suggests that the number of substrate-binding sites may vary with the concentration of the substrate used. It is likely that there is only one substrate-binding site in the lower ranges of substrate concentrations, and there are more than one binding site in the higher ranges; this in turn suggests that there is negative co-

operativity. Picolinic acid hydrazide, the structural analogue of isoniazid, inhibits the Y-enzyme reaction. In the higher ranges of substrate isoniazid concentrations, picolinic acid hydrazide behaves like a competitive inhibitor, whereas the inhibitory pattern does not fit into any particular model at lower concentration of isoniazid. Picolinic acid hydrazide itself, however, cannot undergo the reaction, as for isoniazid, in the presence of NAD (B. Gayathri Devi et al., unpublished work). The enzyme-purification steps used have resulted in a 23-fold increase in specffic activity and a recovery of 50%Y of initial activity. The purified protein is electrophoretically homogeneous (at both acid and alkaline pH) and possesses catalase and peroxidase activities in addition to the Y-enzyme activity. Thermal-inactivation studies and sensitivity to inhibitors and chelating agents also revealed that the three activities are the properties of a single protein. Further, the reaction is stimulated by 02 and the formation of the product is observed in the presence of Mn 2+, non-enzymically (B. Gayathri Devi et al., unpublished work). All these findings point to the fact that the Y-enzyme reaction may be a peroxidatic type of reaction. In that case, it is logical to assume that peroxidases from other sources might catalyse a similar reaction (the Y-enzyme reaction) in the presence of isoniazid and NAD. To test this point, highly purified preparations of peroxidases (horseradish peroxidases, type II and type VI, and lactoperoxidase), from sources vastly different from mycobacteria, were examined. A pure preparation of catalase from ox liver (2 x crystallized) was also tested in the above system. Whereas the peroxidases 1975

PEROXIDASE AND ISONIAZID ACTION

from both plant and animal sources catalyse the Y-enzyme reaction, the ox liver catalase fails to do so. A comparison of the properties of mycobacterial peroxidases and horseradish peroxidase in the Yenzyme reaction was therefore carried out. The mycobacterial enzyme is a high-molecular-weight (240000) protein resembling the catalase preparations, and much different from peroxidase (40000) in size. In contrast, however, the two enzymes show great similarities in their optimal pH requirements and their sensitivity to various classes of inhibitors is the same. On the basis of the findings discussed so far, it is clear that a single mutation from isoniazid sensitivity to isoniazid resistance in M. tuberculosis leads to the loss of isoniazid uptake and a loss of catalase, peroxidase and Y-enzyme activities. The occasional catalase-positive nature of the isoniazid-resistant organism may be satisfactorily explained by a slight modification of the idea put forward by Winder (1964), that some mutations in the gene (that governs the synthesis of this protein which has all the three activities) can lead to the loss of peroxidase and Y-enzyme activity without affecting catalase activity. The mechanism of isoniazid resistance can therefore be summarized as follows: (a) the inability of the organism to take up isoniazid renders it resistant to the drug; (b) concomitant with the development of resistance to isoniazid, the peroxidasecatalase-Y-enzyme activities are lost; (c) peroxidase (Y-enzyme reaction) may therefore be involved in the uptake of the isoniazid. This raises the obvious question of whether, since it is involved in the uptake, the enzyme is located on the membrane. Our results showed that all of the Y-enzyme activity was found in the 1000OOgsupernatant fraction, after sonication of the cells. Previous workers (Darter & Millman, 1957) have shown that the catalase activity in M. tuberculosis H37Rv, as well as in several other species of mycobacteria, is found in the supernatant fractions and seldom in the particulate fractions. On the other hand, this may be due to the easy solubilization of this enzyme during the disintegration of cells, and its membrane localization still cannot be completely ruled out.

We thank the Wellcome Trust, London, for a grant towards equipment, and Dr. Warren Levinson, Visiting Professor from the University of California, San Francisco, for helpful discussions in the preparation of this manuscript.

Vol. 149

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