Biochimica et Biophysica Acta, 1040 (1990) 327-336

327

Elsevier BBAPRO 33744

Functional modification of an arginine residue on salicylate hydroxylase Kenzi Suzuki a n d K u n i h a r u Ohnishi * Department of Chemistry, Faculty of Science, Kanazawa University, Kanazawa (Japan)

(Received 18 December1989)

Key words: Arginine; Dehydrogenation;Glyoxal; Enzymemodification; Salicylatehydroxylase;(P. putida)

Salicylate hydroxylase from Pseudomonas p u t i d a (EC 1.14.13.1, salicylate, NADH:oxygen oxidoreductase) is an FAD-containing monooxygenase, which catalyzes decarboxylative hydroxylation of salicylate to produce catechol in the presence of NADH and 02. By chemical treatment of the enzyme with dicarbonyl reagents, such as glyoxal, the original oxygenase activity was converted to the salicylate-dependent NADH-dehydrogenase activity with free FAD as electron acceptor. One of twenty arginine residues of this enzyme is concerned with this alteration of activity, as shown by the result of its modification at pH 6.9. This result is further supported by the isolation of one arginine-modified enzyme by chromatographic methods on DEAE-Sephadex, A-50 columns. It exhibits the dehydrogenase activity predominantly. This modified enzyme is spectrophotometrically and electrophoretically characterized by a minute conformational change around the active site, and kineticaily by a 7-fold increase in an apparent K m for NADH and a decrease of more than 5-fold in an apparent K m for FAD as electron acceptor, with an apparent Vmx of 22 s - ~ for the dehydrogenase activity. Flow kinetics also showed a marked decrease in the rate for oxygenation of the reduced enzyme-salicylate complex from 21 s - l (native enzyme) to 3.3 s-1 (modified enzyme). These facts suggest that one arginine residue of the enzyme is responsible for the NADH binding site, and chemical modification of one arginine residue of the enzyme induces some conformational change around the active site to alter the catalytic activity from oxygenation to dehydrogenation.

Introduction Salicylate hydroxylase (EC 1.14.13.1, salicylate, N A D H : o x y g e n oxidoreductase [1-hydroxylating, decarboxylating]) isolated from Pseudomonas putida is an FAD-containing monooxygenase consisting of a single polypeptide with a molecular weight of 54000 and catalyzes decarboxylative hydroxylation of salicylate to produce catechol at the expense of N A D H [1-5]. Evidence presented in previous papers has indicated that the enzyme-salicylate complex interacts with N A D H to form an actual reduced intermediate, which reacts with 0 2 to form products [6-9]. This reduced

* Present address: Department of Microbiology,Schoolof Pharmacy, Hokuriku University, Kanazawa, Japan. Abbreviations: CD, circular dichroism; DCIP, 2,6-dichlorophenolindophenol. Correspondence: K. Suzuki, Department of Chemistry, Faculty of Science, Kanazawa University,1-1 Marunouchi,Kanazawa 920,Japan.

intermediate is a true entity to bind and activate oxygen. However, under anaerobic conditions this intermediate transfers electrons to free F A D to produce FADH2. This fact suggests the presence of a binding site of an electron acceptor on this enzyme [3]. In both aerobic and anaerobic reactions, the substrate, salicylate, is clearly exemplified, which accelerates the reduction of the enzyme extremely by lowering the K m value for N A D H [3,9]. These findings would mean the presence of a specific structure of the active site favorable for oxygenation, and prompted us to study some functional changes triggered by modification of its amino acid residue. In this report, we have found that the original oxygenase activity is altered to an N A D H dehydrogenase activity by modification of one arginine residue with dicarbonyl reagents such as glyoxal, 2,3-butanedione and phenylglyoxal. We have further developed a procedure for the isolation of an arginine-modified enzyme after treatment of salicylate hydroxylase with glyoxal, and examined the mechanism for the alteration of the activity kinetically and spectrophotometrically. Part of this work was published previously [10,11].

0167-4838/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)

328 Materials and Methods

Materials FAD, FMN and riboflavin were obtained from Wako Chemicals and purified by gel filtration on a column (1 × 60 cm) of Sephadex G-15, equilibrated and eluted with 30 mM potassium phosphate buffer (pH 7.0) [4]. The compounds used were purchased as follows: 2,3butanedione and 2,4,6-trinitrobenzenesulfonate from Wako Chemicals, glyoxal from Tokyo Kasei Kogyo, phenathraquinone from Nacalai Tesque, N A D H from Kyowa Hakko and phenylglyoxal monohydrate from Aldrich Chemicals. All other chemicals were available from commercial sources and were reagent grade.

Preparation of enzymes Purified salicylate hydroxylase was obtained by the previously described method from P. putida, S-l, grown with salicylate as a carbon and energy source [7]. Aposalicylate hydroxylase was prepared according to the procedure described in Ref. 1. Metapyrocatechase was prepared from P. putida, T-2, by the method of Takemori et al. [12].

Modification of salicylate hydroxylase Salicylate hydroxylase was modified with glyoxal by the method of Nakaya et al. [13]. An amount of 50 ttM salicylate hydroxylase was incubated with 120 mM glyoxal in 0.3 M potassium phosphate buffer (pH 6.9) at 25 ° C or in 0.3 M bicarbonate buffer (pH 9.2) at 0 ° C in the presence of 20 /~M FAD. A fresh solution of glyoxal was added to the enzyme while stirring frequently and the pH of the reaction mixture was not readjusted during the period of incubation. Aliquots of the incubation mixture were removed at intervals and diluted 5-times with cold 7/~M FAD solution to assay the enzyme activities. If it was necessary to separate the incubated protein from other reagents, the sample of preparation was filtered through a Sephadex G-25 column (2 × 40 cm) equilibrated with 30 mM potassium phosphate buffer (pH 7.0). The enzyme was modified with 2,3-butanedione by the method of Marcus [14]. Reaction of 100/xM salicylate hydroxylase and 1 mM butanedione was performed at 25 °C for 3 h in 70 mM boric acid-borax buffer (pH 7.6), containing 20 /~M FAD and 70 mM EDTA in a final volume of 2 ml. After incubation the enzyme was gel-filtered through a column of Sephadex G-25 (1 x 70 cm) equilibrated with 30 mM potassium phosphate buffer (pH 7.0) and then incubated again in the same buffer at 25°C for 2 h. The enzyme was also treated with phenylglyoxal at 25°C for 2 h by the method of Takahashi [15]. The reaction mixture contained 10 /~M enzyme protein, 5 mM phenylglyoxal, 10/~M FAD and 0.1 M potassium phosphate buffer (pH 6.9) in a total vol. of 5 ml. The

number of modified arginine residues in the protein was determined by the change in absorption at 250 nm in the incubated preparations using the molecular extinction coefficient of 11 mM -1 cm-1 for diphenylglyoxalarginine [16]. For modification of lysine residues, 30 tLM apo-enzyme was treated with 2 mM 2,4,6-trinitrobenzenesulfonate in 30 mM potassium phosphate buffer (pH 7.0) at 25 °C in the dark [17]. The modified enzyme was dialyzed against 30 mM potassium phosphate buffer (pH 7.0) to remove the excess reagent. The number of modified residues was determined by the increase of absorbance at 346 nm with the molecular extinction coefficient of 14.5 mM -1 cm -1 in 4% NaHCO 3 solution. Cysteine residues in the enzyme were modified by incubation of 29 ttM salicylate hydroxylase with 160 /~M p-chloromercuribenzoate in 0.1 M potassium phosphate buffer (pH 7.0) for 3 h [18]. The incubated protein was dialyzed against 30 mM potassium phosphate buffer (pH 7.0). The concentration of the reagent was standardized by titrating it with 50/~M glutathione. All other conditions are indicated in the legends of figures and tables.

Isolation of salicylate hydroxylase with one modified arginine residue Salicylate hydroxylase (50 /~M) was incubated with 120 mM glyoxal in 0.3 M potassium phosphate buffer (pH 6.9) containing 20 ttM FAD for 6 h and then precipitated with ammonium sulfate at 70% saturation. The collected protein dissolved in a small amount of 70 mM Tris-HC1 buffer (pH 7.5) was applied to a column of DEAE-Sephadex, A-50 (2.5 × 68 cm), equilibrated with 70 mM Tris-HC1 buffer (pH 7.5) containing 70 mM NaC1 and 2 /~M FAD. Elution of the enzyme was carried out with a linear gradient constructed from 500 ml of 70 mM NaC1 and 500 ml of 140 mM NaC1 in the same buffer system, and then with 500 ml of 140 mM NaC1 buffer system. The NADH-dehydrogenase activities in the eluted fractions (5.7 ml/tube) were monitored and fractions from No. 194 to 210 in Fig. 3 were collected, concentrated with ammonium sulfate as described above and rechromatographed on the same column by the same procedure.

Enzymatic assay NADH oxidation activity of salicylate hydroxylase was measured by a slightly modified method of Ref. 2. The assay medium contained the enzyme, 7 ttM FAD, 100 ttM salicylate and 40/~M N A D H in 1 ml of 30 mM potassium phosphate buffer (pH 7.0). The decrease of absorbance at 340 nm was monitored upon addition of NADH to the assay medium by using a Hitachi model 323 spectrophotometer at room temperature. For glyoxal-modified enzyme, the assay medium contained 60 ttM FAD and 1130 ttM NADH.

329 Oxygenase activity of the enzyme was measured by monitoring a-hydroxymuconic semialdehyde formation at 400 nm by using a molar extinction coefficient of 17 raM-] cm -1 at room temperature in the above assay medium with addition of 0.02 units of metapyrocatechase [19]. Since maximum absorbance at 375 nm of the product was disturbed by a change in the absorption of NADH, the longer wavelength (400 nm) was used. NADH-dehydrogenase activity of the enzyme was obtained by subtraction of the oxygenase activity from NADH-oxidation activity, which was assayed with flavin derivatives as the electron acceptor. The dehydrogenase activities were also determined by changes of absorbance of dichlorophenolindophenol at 610 nm, with the molecular coefficient of 21 mM -~ cm -1, and of potassium ferricyanide at 420 nm, with the molecular coefficient of 1.05 raM-] cm-1. Oxygen uptake by the enzyme was monitored by a Clark electrode with an assay medium containing an appreciable amount of enzyme, 500 #M salicylate and 100 #M NADH in a total vol. of 3.5 ml of 30 mM potassium phosphate buffer (pH 7.0) at 25 ° C. One unit of the enzyme activities is defined as that amount of enzyme which causes the oxidation of 1 #mol of NADH for NADH-oxidation [2], the formation of 1 #mol catechol for oxygenation, the reduction of 1 #mol of electron acceptors, such as dichlorophenolindophenol for NADH-dehydrogenase, or uptake of 1 #mol molecular oxygen for oxygen consumption per minute, at 20 ° C, except for oxygen consumption at 25 ° C. When flavins were used for electron acceptor, a unit of NADH dehydrogenase was calculated from the difference between NADH oxidase and oxygenase activity units observed in an assay system.

Amino acid compositions were determined with a Hitachi model KLA-5 automatic amino acid analyzer. Lyophilized enzyme preparations were hydrolyzed in 6 M HC1 at l l 0 ° C for 24 h in evacuated and sealed vials. Arginine residues in protein were quantitatively analyzed by the Sakaguchi reaction [24] or the phenanthraquinone reaction [25] after acid hydrolysis. Protein was determined by a modified micro-biuret method [26] or Lowry method [27], with crystalline bovine serum albumin as a standard. Results

Isolation of salicylate hydroxylase with one modified arginine residue Salicylate hydroxylase shows oxygenase activity (9098%) with some NADH dehydrogenase activity (2-10%) under ordinary assay conditions. By treatment of the enzyme with glyoxal, the oxygenase activity (10 units/mg of protein, 540 m i n - t ) was decreased and the dehydrogenase activity (0.2 unit/rag of protein, 10 min -1) was increased. Fig. 1 shows changes of the enzyme activity by treatment with 120 mM glyoxal in 0.3 M potassium phosphate buffer (pH 6.9) at 25 °C (Fig. 1A), in which changes of the activity followed along at least two successive exponential processes. The inactivation rate of the oxygenase activity obeyed pseudo-first-order kinetics with respect to the enzyme active site until 85% of the activity was inactivated and prolonged incubation with glyoxal resulted in complete

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E Analytical methods Absorption spectra were measured with a Hitachi model 323 recording spectrophotometer. Circular dichroism spectra (CD) were obtained with the Jasco model L-20 photoelectric spectropolarimeter at room temperature. The molar ellipticity (deg. cm2. dmol -]) is expressed, in the 300-525 nm region, on a molar flavin basis. Stopped-flow experiments were carried out with a Union Giken model RA-1100 stopped-flow spectrophotometer with a 2 mm light path reaction cell according to the general procedure described previously [9]. Native polyacrylamide gel disc electrophoreses were performed in a constant electric current of 2 mA per gel column containing 7.5% acrylamide at 4 ° C according to Davis [20]. Gels were stained with Coomassie brilliant blue R-250 and destained [21]. Disc gel electrophoreses in sodium dodecyl sulfate were carried out by the method of Laemmli [22] with 0.1% sodium dodecyl sulfate and 12.5% acrylamide. Isoelectric focusing analyses were carried out as previously described [23].

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Fig. 1. Inactivation of oxygenase activity and activation of NADHdehydrogenase activity of salicylate hydroxylase by glyoxal. Reactions with 120 mM glyoxal were carded out in 0.3 M potassium phosphate buffer (pH 6.9) at 25°C (A) and in 0.3 M bicarbonate buffer (pH 9.2) at 0 ° C (B). Other conditions were the s a m e a s described under 'Materials and Methods'. Aliquots (100 #1) of the incubation mixture w e r e taken up at the indicated times, diluted with cold 7 #M FAD, and the oxygenase (OX, o) and the NADH-dehydrogenase (DH, t ) activities were assayed. Ratio of the dehydrogenase activity to the sum of the oxygenase and dehydrogenase activities was also plotted (zx).

330 TABLE I

Basic amino acid composition of native and modified salicylate hydroxylase

Data represent mean 5: S.D. for four determinations. Amino acid a

Lysine Histidine Arginine

Residue per mol of protein native enzyme

modified enzyme

18.35:0.3 13.4 5:0.3 20.8 + 0.3

18.65:0.3 13,5 5:0.3 19.6 5:0.3

a Number of amino acid residues per mol of salicylate hydroxylase,

inactivation (not shown). The NADH-dehydrogenase activity enhanced from 10 to 266 rain -1 at the end of the first phase, followed by the decrease on further treatment with glyoxal. The rate constants for the inactivation of oxygenase activity with glyoxal were 4.3. 104 M -1- s -1 for the holt-enzyme and 5.0.104 M -1 • s- ~ for the apt-enzyme in 30 mM potassium phosphate buffer (pH 7.0) at 25 o C. The changes of the dehydrogenase activity by the modification at pH 9.2 is also shown (Fig. 1B). Although glyoxal treatment of the enzyme was extensively carried out at a pH range of 6.0-10.3 *, this pH (pH 6.9) was found to be an optimum for inducing the dehydrogenase activity and for separating the first phase of the inactivation of the oxygenase activity from the second phase. Based on this knowledge about the conditions for glyoxal treatment, we tried the isolation of salicylate hydroxylase with one modified arginine residue from the mixture of enzyme incubated with 120 mM glyoxal in 0.3 M potassium phosphate buffer (pH 6.9) for 6 h. The obtained enzyme preparation after the incubation showed the modification of 1.2 out of 20 arginine residues, and the presence of one major and a few minor discs on the acrylamide gel electrophoresis without sodium dodecyl sulfate. Then, this preparation was chromatographed on a column of DEAE-Sephadex, A-50, as shown in Fig. 2. The dehydrogenase activity induced by modification in the eluted fractions was monitored, and a fraction from No. 194 to 210 in the elution profile in Fig. 2 was collected. This fraction was further rechromatographed on the same column. The protein preparation thus obtained from 100 mg of the native preparation was 30 mg and was homogeneous on polyacrylamide gel electrophoresis.

other basic amino acid residue was affected by modification (Table I). In the analysis, a new ninhydrin-reactive material in the hydrolysates of the modified enzyme was detected in the elution pattern between histidine and ammonia, and the amount was increased in the hydrolysate of protein preparation treated for long hours with glyoxal, indicating the material to derive from arginine residue by modification. Furthermore, the numbers of modified arginine residues were analyzed in the hydrolysates of the native and modified enzyme; 20.1 + 0.3 (native) and 19.1 ± 0.3 (modified) by Sakaguchi reaction, and 20.3 ± 0.3 (native) and 19.3 + 0.3 (modified) by phenanthraquinone reaction, supporting the results as described above. When the enzyme was treated with 120 mM glyoxal in 0.3 M potassium phosphate buffer (pH 9.2), the original oxygenase activity was decreased to 37 and 4%, and the numbers of modified arginine residues were 4.4 and 10.1 with concomitant loss of 0.4 and 2.5 lysine residues, for 3 and 24 h incubation, respectively. The numbers of other amino acids did not change. These findings confirm the preservation of other basic amino acid residues except for the arginine residue in the modified enzyme obtained above.

General properties of the modified enzyme Electrophoretical behaviors of the native enzyme and the modified enzyme did not differ significantly in polyacrylamide gels in either the presence or absence of sodium dodecyl sulfate. Enzyme preparations with more than two modified arginine residues had larger mobilities in gel without sodium dodecyl sulfate. Isoelectric focusing experiments gave pI of 5.0 for the native enzyme and of 4.9 for the modified enzyme.

E OQ8 OO c

Amino acid analysis of the modified enzyme indicates that only one of twenty arginine residues but no

* The rate of reaction between this enzyme and glyoxal is remarkably dependent on pH; as pH increase, the rate increases. The inflection point is near pH 8.7.

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Fig. 2. The first fractionation of glyoxal-modified salicylate hydroxylase on a DEAE-Sephadex, A-50, column (2.5 x 68 cm). Conditions of the enzyme modification and its fractionation were described in the text. Each fraction (5.7 ml) was assayed for the NADH dehydrogenase activity (o) and the absorbance at 280 nm (0). Ratio of the dehydrogenase activity to the absorbance were calculated and plotted (A). Fractions from No. 194 to 210 were combined as indicated by the bar and concentrated.

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Fig. 3. CD-spectra of native and modified salicylate hydroxylase in the presence and absence of salicylate. CD-spectra were measured with 50 /~M enzyme preparation in 30 m M potassium phosphate buffer (pH 7.0). Spectrum 1 ( ), native enzyme; 2 ( . . . . . ), modified enzyme; 3 (. . . . . . ), native enzyme and 1 m M salicylate; 4 (. . . . ), modified enzyme and 1 m M salicylate.

Fig. 3 shows CD spectra of the native and modified enzymes in the region of 300-530 nm. Peaks of the modified enzyme (curve 2) and its complex with salicylate (curve 4) shifted to a longer wavelength by 2-3 nm than those of the native enzyme (357 nm) (curve 1) and its complex (350 nm) (curve 3), respectively. A large difference was observed between the spectra of the native (curve 3) and modified (curve 4) enzymes complexed with salicylate in the ellipticity of the respective peaks. The two enzyme preparations had similar absorption spectra in the region of 340-600 nm. Like the CD spectra, the absorption peak of the modified protein around 375 nm shifted slightly to a longer wavelength. The shift was also observed with its salicylate complex. The dissociation constants of the enzyme-salicylate complex were determined by spectrophotometric titration at 480 nm to be 3.5 and 4.0/xM for the native and modified enzyme, respectively. Effects of FAD and electron acceptors on actioities of the native and the modified enzymes The low concentrations of FAD caused only oxygenase activity to both native and modified apo-enzyme preparations in the presence of the substrate, salicylate, without NADH-dehydrogenase activity (Fig. 4A and B). By further excess additions of flavin, the dehydrogenase activity was induced on the modified enzyme preparation (Fig. 4B). The activity was further increased with concomitant decrease of the oxygenase activity at a higher concentration of FAD (Fig. 4D). With the native enzyme, additions of a large amount of FAD (more than 50/~M) were necessary to induce the dehydrogenase activity (Fig. 4C). In the presence of

other substrates, 2,3-dihydroxybenzoate, 2,5-dihydroxybenzoate or p-aminosalicylate, similar behaviors on both activities were observed by titration of apo-enzyme with FAD. On the other hand, NADH-oxidation activity observed in the presence of a pseudosubstrate, benzoate [9], was hyperbolically reconstituted by addition of FAD into both apo-enzyme preparations. By addition of higher concentrations of F A D up to 50 #M, the activity of NADH oxidation was biphasically increased with the modified enzyme but not with the native enzyme. Under these conditions, oxidation of NADH was negligible with both enzyme preparations without substrate or pseudosubstrate. From these results it could be concluded that with up to the equivalent amount of FAD, both enzyme preparations act as the oxygenase, but excess FAD induces the dehydrogenase activity to both enzyme preparations with different affinities for FAD; the modified enzyme with strong affinity and the native enzyme with weak affinity. FMN, riboflavin and others listed in Table II played a role of electron acceptors to emerge out of the dehydrogenase activity of the enzyme preparations. Similarly, with the dehydrogenase reaction with FAD as an I

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Fig. 4. Effect of F A D on the oxygenase activity and NADH-dehydrogenas¢ activity of native and modified salicylate hydroxylase. Native apo-enzyme (40 nM), (A, C) and modified apo-enzyme (20 nM), (B, D) were used for assay of the enzyme activities. (A) and (B) demonstrate the results of experiments at the low concentration of F A D and (C) and (D) at the high concentration of FAD. (OX, o), the oxygenase activity and (DH, O), the NADH-dehydrogenase activity. Other conditions were shown in the text.

332 T A B L E II

Effects of electron acceptors on NADH-dehydrogenase activity of salicylate hydroxylase Assay mixture contained 20 n M native or glyoxal-modified salicylate hydroxylase, 3 ~ M FAD, 100/zM salicylate, various concentrations of electron acceptors and 100 ~ M N A D H in 30 m M potassium phosphate buffer (pH 7.0), and was incubated at room temperature. Apparent K m values were determined by double reciprocal plotting the dehydrogenase activities vs. the concentrations of aeceptors or N A D H . Apparent Vn~x values were calculated from the extrapolation of N A D H concentration on those plots. Acceptor

Native

Modified

K m (/~M) for

Vm~,

K m (/~M) for

Vm~x

accep-

(rain-l)

accep- N A D H

(min-l)

NADH

tor FAD > 100 3.9 FMN 20 9.7 Riboflavin 55 5.7 Ka[Fe(CN)6 ] 56 17 DCIP 8.7 19

tor 1 360 1120 860 5500 690

20 7.9 9.3 33 1.2

29 28 28 63 38

1 320 930 900 3900 950

electron acceptor, apparent K m values of the modified enzyme for these acceptors were smaller than the respective values of native enzyme and the opposite relation was observed for an apparent K m value of N A D H . By the decrease of 02 concentration from 257 to 0 ~tM, the dehydrogenase activity in the presence of FAD (5-10/~M), 2,6-dichlorophenolindophenol or potassium ferricyanide in 30 m M potassium phosphate buffer (pH 7.0) increased in the native enzyme, and increased only slightly in the modified enzyme. The dehydrogenase activity was competitively inhibited by 02 ( K i = 47/~M) and oxygen uptake was also competitively inhibited by potassium ferricyanide ( K i = 5 0 / x M ) with the native enzyme.

Steady-state and stopped-flow kinetics Apparent K m values for F A D as the prosthetic group and substrates of oxygenase activity, in the native and modified enzymes, are shown in Table III. Chemical modification of the enzyme brought about a large change of an apparent K m value for N A D H but not significantly for others, and decreased the apparent maximum velocity of the oxygenase activity to a large extent. From the double reciprocal plots of velocity vs. 02 concentration, the maximum velocities of the oxygenase activity were determined to be 730 and 180 min-1 for the native and the modified enzymes, respectively, in the absence of free FAD. With a higher concentration of FAD, the oxygenase activity of the modified enzyme decreased concomitantly with an increase of the dehydrogenase activity. While the oxygenase activities of both native and modified enzyme preparations had an optimum p H around 7.0, the dehydrogenase activity of the modified enzyme increased at a more acidic p H region tested (pH 4.7-6.0).

The reaction mechanism for the native enzyme was previously analyzed by stopped-flow apparatus [9]. The similar analysis was carried out for the modified enzyme. The enzyme-salicylate complex formation was analyzed in the presence of the large excess of salicylate by monitoring the absorbance at 480 nm, characteristic of the complex. The rate constants for formation and dissociation of the complex, k t (1.2.107 M - 1. s - 1) and k_ 1 (48 s - l ) , respectively, were slightly smaller than the respective values for the native enzyme. The reduction of the enzyme-salicylate complex with N A D H and the reoxidation of the reduced enzyme-salicylate complex were monitored by the absorptional change at 450 nm. The reciprocal values of their pseudo-first-order rate constants were plotted against the reciprocal values of the concentrations of N A D H or 02, giving linear relationships between 1/k vs. 1 / N A D H or 1/O2; the intercepts on the ordinate were not zero. From the intercepts, the maximum rate constants, k 2 for the reduction and k 3 for the reoxidation were determined to be 140 s -1 and 3.3 s -1, respectively. As shown in Table IV, it is interesting that the rate for reoxidation of the reduced enzyme-salicylate complex was enormously effected by glyoxal modification; 3.3 s -1 for the modified enzyme as compared with 21 s -1 for the native enzyme. Kinetic intermediates, X 1 and X 2 were assumed for the reduction and the reoxidation, respectively. Though X~ was not characterized spectrophotometrically, the dissociation constant, 23 ~tM was determined from the intercept on the abscissa, which is larger than the value for the native enzyme, 7.9 /~M. Similarly, the dissociation constant, 125 /~M was estimated for the intermediate of the reoxidation, )(2, and is a little larger than the corresponding value, 110/aM, for the native enzyme. X 2 seemed to be the flavin(4a)hydroperoxide [28] from its spectrum with absorption maximum around 375 nm (unpublished data, T A B L E III

Kinetic constants of native and modified salicylate hydroxylase for oxygenase activity Enzyme concentrations in the assay systems were 40 n M for the native enzyme and 20 n M for the modified enzyme. In the case of determining K m for FAD, apo-enzyme preparations were used. Apparent K m values were determined by double reciprocal plotting the oxygenase activities vs. the concentration of F A D or substrate. Apparent Vm,x values were calculated from the extrapolation of oxygen concentration on those plots. Native enzyme

Modified enzyme

K m (/~M) FAD a Salicylate NADH 02

0.12 1.6 3.9 110

0.12 0.73 29 110

Vmax (min - 1)

730

180

a As prosthetic group.

333 TABLE IV

where S and P denote salicylate, and products, catechol, CO2 and H20 , respectively, and X1 and X2 kinetically required intermediates.

cation e x p e r i m e n t s of salicylate hydroxylase with other d i c a r b o n y l reagents, such as 2 , 3 - b u t a n e d i o n e a n d p h e n ylglyoxal. W h e n the e n z y m e was treated with 1 m M b u t a n e d i o n e at 25 o C for 3 h, i n 70 m M boric a c i d - b o r a x buffer ( p H 7.6), 90% loss of the oxygenase a n d a 2 - 3 fold increase of N A D H - d e h y d r o g e n a s e activity were observed. These altered activities were almost completely reversed b y r e m o v a l of the residual reagent a n d borate, followed b y i n c u b a t i o n at 25 o C for 2 h. I n c u b a tion of the hydroxylase with 5 m M p h e n y l g l y o x a l i n 0.1 M p o t a s s i u m p h o s p h a t e buffer ( p H 6.9), at 25 ° C for 2 h, b r o u g h t a b o u t a loss of 97% oxygenase activity, b u t more t h a n a 10-fold increase in the rate of the d e h y d r o genase to the s u m of the d e h y d r o g e n a s e a n d oxygenase activities, c o n c o m i t a n t l y with m o d i f i c a t i o n of 0.95 arginine residue. Because d i c a r b o n y l reagents have b e e n reported to react n o t only with a r g i n i n e b u t also with lysine [13,15,30] or cysteine [30] residue, we investigated the possibility of these residues p a r t i c i p a t i n g in the conversion of activities. M o d i f i c a t i o n of two residues of lysine b y 2,4,6-trinitrobenzene sulfonate or 2 - 3 residues of cysteine b y p - c h l o r o m e r c u r i b e n z o a t e resulted in the respective 71 or 67% loss of the original oxygenase activity, b u t n o e n h a n c e m e n t of the d e h y d r o g e n a s e activity. Therefore, these residues m a y be involved in the role of the oxygenase f u n c t i o n b u t n o t in the c o n v e r s i o n of the activities.

Rate constant

Native enzymea

Modifiedenzyme

Discussion

k 1 (M-l-s -1) k_ 1 (s -1) k 2 (s - l ) k 3 (s -1)

1.8.107 62 230 21

1.2.107 48 140 3.3

Kinetic parameters for the partial reactions of native and modified salicylate hydroxylase These analyses were carried out according to the general procedure described previously [9]. Rate constants were measured in 30 mM potassium phosphate buffer (pH 7.0) at 25°C. Formation of the enzyme-saiicylate complex (EFS) was monitored by measuring the increase of absorbance at 485 nm at different times from 1.1 ms to 2.0 ms after mixing 20 #M enzyme with an equal vol. of 200 #M (large excess) salicylate in flow-apparatus. For estimation of the rate of binding of salicylate to the enzyme (kl) and the rate of the reverse reaction (k_l), the dissociation constant of the enzyme-salicylate complex, 4.0 #M was used for the modified enzyme. Rate constant for the reduction of the enzyme-salicylate complex was estimated from the change of absorbance at 450 nm after mixing 10 #M enzymes with 40-200 #M NADH anaerobically. Both syringes contained 100 #M salicylate. The reoxidation of reduced enzyme-salicylate complex (EFH2S) was monitored by measuring the increase of the absorbance at 450 nm after mixing 10 #M reduced enzyme-salicylate complex with 60-257 #M 02 solutions in the presence of 100 #M salicylate. The rate constants, k 1, k_ 1, k 2 and k3, are for the formation of enzyme-salicylatecomplex (EFS), the dissociation of the complex, the reduction of EFS with NADH and the reoxidation of EFH2S with oxygen, respectively: S NADH 2 \k~ \ • EFS. "X~ EF.

O~ k2

" EFH2S.

")(2

k-i k3

"EF+P

The present results established o n e a r g i n i n e residue as a f u n c t i o n a l group i n the o x y g e n a t i o n catalyzed b y salicylate hydroxylase. T h e chemical m o d i f i c a t i o n of

a The rate constants for the native enzyme were quoted [9]. TABLE V Suzuki, K.).

Protection of oxygenase activity f r o m glyoxal inactivation Substrates, p s e u d o s u b s t r a t e or N A D H protected the oxygenase activities of the apo- or h o l o - e n z y m e from glyoxal i n a c t i v a t i o n effectively (Table V). F A D protected the a p o - e n z y m e o n l y slightly b u t did n o t protect the h o l o - e n z y m e against i n a c t i v a t i o n of the oxygenase activity by glyoxal. Significant p r o t e c t i o n against the i n a c t i v a t i o n was p r o v i d e d b y 3-methylsalicylate, salicylate, b e n z o a t e a n d even more b y N A D H . C o m b i n a t i o n of N A D H a n d salicylate protected the a p o e n z y m e almost completely against the inactivation.

Chemical modification o f salicylate hydroxylase with other reagents T h e p a r t i c i p a t i o n of o n e a r g i n i n e residue for the a l t e r a t i o n of the oxygenase activity to the dehydrogenase activity was further s t r e n g t h e n e d b y the modifi-

Effects of substrates or cofactors on reactivation of oxygenase activity of holo- and apo-salicylate hydroxylase by glyoxal Holo-enzyme (10 #M), or apo-enzyme (1 #M) of salicylate hydroxylase, was incubated with 0.8 M glyoxal and various concentrations of substrate or cofactor as indicated in a final vol. of 2 ml of 0.3 M potassium phosphate buffer (pH 6.9) at 25 o C, containing 7 #M FAD only for holo-enzyme. Aliquots (0.2 ml) of the incubation mixtures were withdrawn at various time intervals, mixed with 0.8 ml of 7/~M ice-cold FAD solution and assayed for oxygenase activity. Addition None FAD, 50/tM Salicylate, 1 mM 3-Methyisalicylate, 1 mM Benzoate, 1 mM NADH, 2 mM NADH, 20 mM NADH, 2 mM + salicylate, 1 mM n.d., not determined.

Half-time of inactivation (min) holo-enzyme

apo-enzyme

29 26 170 296 53 n.d.a n.d. n.d.

17 24 60 122 42 20 187

334 this arginine residue with glyoxal caused the enzyme to act as dehydrogenase but not as oxygenase. That this alteration of activity is due to the modification of an arginine residue on the enzyme would be exemplified as follows. Not only glyoxal but also other arginine reagents such as 2,3-butanedione and phenylglyoxal converted the activity of the enzyme to the dehydrogenasea~ivity. The experiments on the reversible conversion of activities by butanedione treatment could be considered as confirmation for participation of an arginine residue. As dicarbonyl reagents have, however, been reported to modify not only arginine but also lysine [13,15,30] or cysteine [30] residues, the modification of these residues by the respective chemical reagents, 2,4,6-trinitrobenzenesulfonate and p-chloromercuribenzoate, was carried out. It, however, resulted in the loss of oxygenase activity but not the enhancement of dehydrogenase activity. The modification of only one arginine residue would be sufficient for the conversion of the activity of the enzyme from the following evidence. The dehydrogenase activity increased for 6 h and then kept constant under the modification of salicylate hydroxylase with 120 mM glyoxal at p H 6.9 (Fig. 1). As the modified arginine residue of the modified enzyme after 6 h incubation was 1.2 residue, the enhancement of the dehydrogenase activity would be due to only one arginine residue. The modification of more than 2 arginine residues may bring about the decrease of the enhanced dehydrogenase activity (Fig. 1B). The same conclusion was further obtained by development of the dehydrogenase activity by 0.95 arginine-modified enzyme treated with phenylglyoxal. In an attempt to elucidate the role of an arginine residue on salicylate hydroxylase in details, it is necessary to isolate a species of the modified enzyme on which one arginine residue is changed. This was accomplished by fractionation of the modified enzymes obtained with glyoxal treatment under the mild conditions, through DEAE-Sephadex columns (Fig. 2). This purified modified enzyme was homogeneous on disc gel electrophoresis which can discriminate the degree of arginine modification on the enzyme by its mobility; the higher the degree of modification the larger its mobility. The number of modified arginine residues on the enzyme was determined by amino acid analyzer (Table I) and by specific methods for arginine residues, such as Sakaguchi and phenanthraquinone reactions, which were 1.2, 1.0 and 1.0 of twenty arginine residues, respectively. These facts indicate that this isolated preparation got one arginine residue modified. That the activity stimulated by modification is dehydrogenase activity but not oxidase activity was undoubtedly demonstrated by the findings that N A D H oxidation was not affected in the absence of oxygen, and was not observed in the absence of free FAD or

other electron acceptors even in the presence of oxygen. Therefore, this activity is different from the oxidase activity observed by salicylate hydroxylase [31,32] and p-hydroxybenzoate hydroxylase [33] in the presence of a 'pseudosubstrate', such as benzoate. The titration of apo-salicylate hydroxylase with F A D showed that the oxidase activity in the presence of benzoate was enhanced hyperbolically even in the absence of free FAD. Under the standard assay conditions which contained 7 btM FAD, the oxygenase and dehydrogenase activities were 541 and 10 min -1, respectively, for the native enzyme, and 74 and 266 min-1, respectively, for the modified enzyme. From kinetical analysis (Table II and III), these altered activities were due to a decreased g m for FAD as electron acceptor, 20 /~M, and a decreased Vmax for oxygenation, 180 min -1, as compared with those of the native enzyme, more than 100/~M and 730 min-1, respectively. As the ratio of the oxygenase to dehydrogenase activity for the modified enzyme depended on F A D (and 02) concentration, the addition of the excess concentration of FAD could exhibit only the dehydrogenase activity with a Vm~ of 1320 rain -1 Other kinetic parameters were not greatly affected by modification, except for K m for N A D H from 3.9 /~M (native) to 29/tM (modified). Flow kinetics has shown a remarkable decrease in the rate constant for oxygenation of the reduced enzyme-salicylate complex (EFH2S) with 02 from 21 (native) to 3.3 s -1 (modified) (Table IV). This change appears to correspond with a decrease of Vm~x for oxygenation from 730 to 180 min -1 in the absence of free FAD (Table III). These kinetic results would mean that the altered properties of the modified enzyme are due to the altered binding ability for electron acceptor and the altered reactivity with 02, which may be induced by the conformational change around the active site of the enzyme, as described later. Therefore, a direct change of the enzyme by modification with glyoxal may be relevant to a change in oxidation-reduction properties of the prosthetic group. Based on previous knowledge and results obtained from the present work, a relation between the oxygenase and the dehydrogenase reaction catalyzed by salicylate hydroxylase would be described as follows: EF.

S

NADH;z

" EFS. • EP + P

EF.

S

"X

~ EFH2S.

02 " [EFH2SO2]

Oxygenation

NADH2

"EFS.

• EFS + AH2

"X

~ EFH2S.

A

" [EFH2SA]

Dehydrogenation

where E, E F H 2, S, A, P and X mean the oxidized and reduced forms of the enzyme, substrate, electron acceptor, products (catechol, CO 2 and H 2 0 ) and kinetic intermediate, respectively. The active intermediate, EFH2S , which should react 02 in the native enzyme,

335 appears to be changed to an inactive or unfavorable form as an intermediate of oxygenation in the modified enzyme, so followed by donating electrons to acceptor. This dehydrogenation is observed anaerobically [6], and partially even in aerobic conditions [6] or with exogenous F M N or flavin analogs [34,35]. This arginine residue would be located at the active site or N A D H - b i n d i n g site, since K m for N A D H was greatly increased by the modification and the protection against the alteration of activity by glyoxal was observed by salicylate and even more by N A D H . A combination of N A D H and salicylate protected almost completely against this conversion (Table V). These effects suggest this arginine residue location as an N A D H binding site. In p-hydroxybenzoate hydroxylase, one arginine residue is involved in the substrate binding as the result of chemical modification with phenylglyoxal [36], which agrees well with the existence of a salt bridge between the carboxylate group of substrate and the guanidium group of Arg-214 [37]. In addition, two other arginine residues have been suggested to participate in the N A D P H binding site [38]. However, this appears to be insufficient to explain the increase in the affinity for the electron acceptors and the decrease in the reactivity with O z, since the modified enzyme has only one arginine residue modified. The modification of the arginine may not only change the interaction between the enzyme and N A D H , but also, or as a result of this, produce a conformational change of the active site, leading to the effects on the K m values. This possibility could well be reconciled with the altered kinetic properties described above. The conformational change will be aroused not grossly but limitedly near the active site of the modified enzyme. This seems to be a reasonable assumption from the minute difference of C D (Fig. 3) and absorption spectra of the enzymes at visible region and a decrease of pI from 5.0 (native) to 4.9 (modified). Electrophoretic behaviors of both enzymes did not differ in polyacrylamide gel in either the presence or absence of sodium dodecyl sulfate. This study further demonstrated that under aerobic conditions, the native enzyme can also exhibit the dehydrogenase activity in the presence of a large excess concentration of F A D (more than 50/~M), other flavins, F M N and riboflavin which could not be substituted for F A D as a prosthetic group of the enzyme [2], an artificial electron acceptors such as 2,6-dichlorophenolindophenol and potassium ferricyanide (Table II). An FAD-containing oxygenase cleaving 3-hydroxypyridine ring has recently been reported to have a partial dehydrogenase activity under the standard assay conditions [34]. These facts suggest that the intermediate, the reduced enzyme-salicylate complex should be formed only under much specialized conditions in order to potentiate the following oxygenation. If these conditions favorable

for oxygenation are not sufficiently established as optim u m assay conditions or disturbed by such as the alteration of the structure around the active site of the enzyme, this intermediate will decay to give its electrons into the acceptors. The original oxygenase activities of other flavin-containing monooxygenases, such as imidazoleacetate monooxygenase [39] and lysine monooxygenase [40] have been altered to the oxidase activities by the chemical modification of their cysteine residues. These conversions of activity will be explained by the idea described above. Our conclusion is that an arginine residue at the active site of salicylate hydroxylase not only involves itself in the N A D H binding, but also plays an essential role in creating an active or specialized conformational state of the reduced enzyme-salicylate intermediate for oxygenation.

Acknowledgements This investigation was supported in part by research grants from the Ministry of Education of Japan. We are indebted to S. Riku, T. Suzuki, Y. Hara, Y. Toyoda, M. Kitamura, K. Tani, M. Sugisawa and K. K a w a b a t a in this laboratory for their technical assistance. The authors gratefully acknowledge the generous advice received from Dr. M. Katagiri and Dr. E. Itagaki of our laboratory.

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