- Email: [email protected]
The reaction of chicken liver sulfite oxidase with dimethylsulfite
Biochi~ic~a
ELSEVIER
et Biophysica A~ta Biochimica et Biophysica Acta 1253 (1995) 133-135
Rapid Report
The reaction of chicken liver sulfite oxidase with dimethylsulfite Michael S. Brody, Russ Hille * Department of Medical Biochemistry, The Ohio State University, 1645 Neil Avenue, 333 Hamilton Hall, Columbus, OH 43210-1218, USA Received 18 August 1995; accepted 28 August 1995
Abstract
We have undertaken a steady-state and rapid kinetic study of the reaction of enzyme with sulfite and dimethylsulfite. Methylation of sulfite results in a sign:ificant increase in K m and Kd for the substrate in the course of steady-state and rapid reaction kinetics, respectively, but kcat and the limiting rate constant for enzyme reduction (kred) are essentially unchanged. This indicates that while substrate oxyanion groups are effective in stabilizing the Eox. S complex, the breakdown of this complex proceeds at the same rate even in their absence. The critical element of the substrate required for reactivity is a suitable lone-pair available to undertake nucleophilic attack on a Mo=O group of the active site. Keywords: Oxo molybdenum enzyme; Enzyme reaction mechanism; Metalloenzyme
Sulfite oxidase (EC 1.8.3.1) catalyzes the oxidation of sulfite to sulfate with subsequent reduction of two equivalents of cytochrome c in the process; the oxygen atom incorporated into substrate is ultimately derived from water. The enzyme is one of the better understood members of the oxo molybdenum class of enzymes possessing a Mo w 0 2 unit in the active site [1], and the fundamental chemistry catalyzed by these enzymes has been investigated using several different model systems possessing a MoVIO2 unit [2-9]. These model compounds possess the ability to catalyze oxygen atom transfer between an oxo donor and acceptor by cycling between LnMoVlO2 and LnMoWO species, and the suitability of a given donor:acceptor pair for effective turnover has been adequately justified on thermodynamic grounds [4]. Phosphines have proven to be particularly useful as oxo acceptor species, reacting with LnMoWO2 to give LnMolVO and the corresponding phosphine oxide. Based on the negative activation e~Ltropies observed for reactions of the type MoWO2 + :PR 3 --~ MolVO + OPR 3, it has been concluded that the reaction proceeds via an associative transi-
* Corresponding author. Fax: + 1 (614) 292 4118. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 5 ) 0 0 1 9 4 - 8
tion state formed by nucleophilic attack of the phosphine lone pair on one of the M o = O groups [7]:
C. OC-':PR
[ •. LMolV=O + O=-PR3
At least one oxo molybdenum enzyme, dimethylsulfoxide reductase from Rhodobacter sphaeroides, is able to use the water-soluble phosphine 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane as a reducing substrate in a reaction directly analogous to that in which the model compounds participate [10]. These observations emphasize the strong analogy between the chemistry of the model compounds and enzymes. The reaction of sulfite oxidase with sulfite might reasonably be expected to proceed in an analogous fashion to that proposed for the phosphine in the model compound chemistry, with the sulfur lone pair of sulfite undergoing nucleophilic attack on a M o = O group. Alternatively, it has been suggested that the catalytic sequence of sulfite oxidase begins with direct coordination of sulfite to the molybdenum via one of the oxyanion groups [11]. This view is consistent with the known ability of a variety of anions to inhibit sulfite oxidase [12-15], and to perturb the
134
M.S. Brody, R. Hille / Biochimica et Biophysica Acta 1253 (1995) 133-135
Mo v EPR signal of partially reduced enzyme [16-21]. In an effort to gain further insight into the reaction mechanism of sulfite oxidase, we have undertaken a steady-state and rapid kinetic study of the reaction of enzyme with sulfite and dimethylsulfite (the rate of hydrolysis of dimethylsulfite in water is 10 -5 s -1 [22]). We find that while methylation of sulfite results in a significant increase in K m and K 0 for the substrate in the course of steady-state and rapid reaction kinetics, respectively, kcat and the limiting rate constant for enzyme reduction (kr¢0) are unchanged. These results indicate that while substrate oxyanion groups are effective in stabilizing the Eox • S complex, the breakdown of this complex proceeds at the same rate even in their absence. We conclude that the critical element of the substrate required for reactivity is a suitable lone-pair available to undertake nucleophilic attack on a M o : O group of the active site. Steady state analysis following the catalytic velocity of sulfite oxidase J as a function of the sulfite and dimethylsulfite concentration under conditions of excess cytochrome c gave typical hyperbolic plots. A kca t of 79 S- 1 and a K m of 28 ~ M are found for sulfite, the K m in good agreement with published results [26]. The corresponding values obtained using dimethylsulfite are 89 s - ' for kca t and 6.3 mM for Kin. The close agreement of kcat for sulfite and dimethylsulfite suggests that breakdown of the Eox. S complex is not affected by substrate methylation; instead, the principal effect of methylation is on Km. The present results give a value for k c a t / K m of 2.8 X 10 6 M -1 s - I with sulfite and 1.4. 10 4 M -1 s - ! with dimethylsulrite. In order to gain greater insight into the reaction of sulfite and dimethylsulfite with sulfite oxidase, the reaction of enzyme with the two substrates has been examined by stopped-flow spectrophotometry, using the absorbance
i Sulfite oxidase was purified by a previously described method [23], with some modifications, involving homogenization of fresh chicken livers in liquid nitrogen, followed by centrifugation, ammonium sulfate fractionation and sequential chromatography on DEAE-Sepharose, Sephacryl-S-200, and a phenyl-Sepharose column. Damage to the molybdenum center was reduced by avoiding an acetone extraction in this procedure. The purified enzyme possessed a heme to protein absorbance ratio (A 414//A 280) o f > 0.5 and specific activity of 2.1- 103 ~ m o l / s - mg. Unless otherwise stated, all experiments were performed at 250(2 in 20 mM Tris adjusted to pH 8.0 with acetic acid, containing 2 mM Na2MoO 4 and 0.1 mM EDTA. The steady state experiments were performed aerobically using a saturating concentration of cytochrome c, 21 /xM (10-fold greater than K m) [24] and varying the concentration of either sulfite or dimethylsulfite. Rates were determined by following the reduction of cytochrome c at 550 nm [25], using an extinction change of 19 630 M - i c m - l . This emperically determined number was used because of the lower spectral resolution of the diode array spectrophotometer used in the present work. Cytochrome c [horse heart, (96% pure)] was obtained from Sigma. Dimethylsulfite ( > 99% pure) was obtained from Aldrich. All other chemicals were of reagent grade and used without additional purification.
0.10
A 0.08
0.06 ,.o O 0.04
0.02
0.00 0.0
!
I
0.1
0.2
i
I
i
0.3
0.4
1/[Sulfite] (uM) 0.10
0.08
0,06 O m
0.04
0.02
O.OG
.
0.0
.
.
.
~
0.5
.
.
.
.
J
.
1.0
.
.
.
1.5
1/[dimethylsulfite] (raM) Fig. 1. Double reciprocal plots for the reaction of sulfite oxidase with sulfite (Panel A) and dimethylsulfite (Panel B). Sulfite oxidase (0.4/zM in the reaction with sulfite, 0.5 #,M in the reaction with dimethylsulfite) was reacted with varying concentrations of substrate in a stopped-fiow apparatus, with the reaction monitored spectrophotometrically at 426 nm. The kinetic constants kred and K d, obtained from the reciprocal of the y-axis intercept and negative of the reciprocal of the x-axis intercept, respectively, are 194 s - 1 and 3 3 / z M for sulfite, and 170 s - i and l I mM for dimethylsulfite. Reaction conditions were 0.02 M tris-acetate buffer ( p n 8.0), 25°C.
change of the enzyme heme as a spectroscopic probe. 2 The reduction of oxidized sulfite oxidase at pH 8.0 by sulfite or dimethylsulfite under anaerobic conditions exhibits well-behaved single-exponential kinetics at all substrate concentrations which are independent of the wavelength monitored. Double reciprocal plots of the observed rate constant vs. substrate concentration for the reaction of enzyme with sulfite (Fig. 1A) and dimethylsulfite (Fig. 1B) are linear, giving a kred of 194 s -1 and a K d of 33
2 Rapid kinetics experiments were performed anaerobically with fully oxidized enzyme using methods described previously [27]. In the present analysis, it is assumed that intramolecular electron transfer from the molybdenum center to the heme of sulfite oxidase is as rapid as was seen in a related study by Sullivan et al. [28], and rate-limited by the chemistry taking place at the molybdenum center.
M.S. Brody, R. Hille / Biochimica et Biophysica Acta 1253 (1995) 133-135
/zM for sulfite; the corresponding values for dimethylsulrite are 170 s-1 and 11 mM. A comparison of the kre d values for the two substrates indicates that this rate constant, which represents the breakdown of the Eox • S complex to Ered + P, is essentially unaffected by methylation of the substrate, consistent with the steady-state results. Methylation of substrate results in a 300-fold increase in K d, indicating a sub:~tantial reduction in the affinity of enzyme for substrate. Overall, these results indicate that while the oxyanion :groups of sulfite are important for substrate binding, perhaps by direct coordination to molybdenum, they are not required for catalysis. A comparison of i~ca t and kre d for sulfite (79 s-1 and 194 s -1, respectively) indicates that the later value is substantially larger. This implies that the reaction of oxidized enzyme with stllfite (or dimethylsulfite) is not ratelimiting in overall turnover for sulfite oxidase and that reaction of reduced enzyme with cytochrome c in the oxidative half of the catalytic cycle must contribute significantly in limiting turnover rate. The oxo molybdenum enzymes may be subdivided into Mo w 02- and Mo v~OS-containing categories [ 1]. The latter group of enzymes, with the exception of molybdenum-containing CO dehydrogenase from species such as Pseudomonas carboxydo~orans, catalyze hydroxylation reactions at carbon centers that necessarily entail cleavage of a C - H bond. The sulfido group is essential for this reaction, as its replacement with a second M o : O group (by reaction with cyanide) renders these enzymes inactive, and it presumably plays a direct role in the cleavage of the C - H bond. The MoVIO2 group of enzymes, including sulfite oxidase, catalyzes a possibly more facile oxygen atom transfer into or out of heteroatom centers (nitrogen, sulfur or arsenic). These re~.ctions invariably involve the reaction of oxidized enzyme with an oxo acceptor possessing a lone electron pair (e.g., sulfite oxidase) or reduced enzyme with an oxo donor to give a product that possesses a lone pair (e.g., nitrate reductase or DMSO reductase). In the case of sulfite oxidase, the present results indicate that it is the availability of a lone pair capable of accepting an oxo group that is the principal element determining suitability as a substrate for tJae enzyme; methylation of the two oxyanion groups reduces the affinity of substrate for the active site, but once the Michaelis complex is formed (at sufficiently high substrate concentration) it breaks down with the same rate ctmstant as does sulfite. In the absence of steric effects due 1:o methylation, the large difference in K d observed for sulfite and dimethylsulfite indicates that substrate oxyanions contribute 3.4 kcal/mol toward the stabilization of the l~.ox • S complex, consistent with direct coordination to the molybdenum (although participation in an electrostatic or hydrogen bonding interactions with other functional groups of the active site cannot be excluded). Dimethylsulfite does exhibits saturation kinetics,
135
however, implying that a pre-equilibrium (Michaelis) complex is formed with this substrate and that the interactions between substrate and active site other than those involving oxyanions must also contribute to stabilization of the complex.
Acknowledgements This work was supported by National Institute of Health grant number AR38917.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
[15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28]
Hille, R. (1994) Biochim. Biophys. Acta 1184, 143-169. Holm, R.H. and Berg, J.M. (1986) Acc. Chem. Res. 19, 363-370. Holm, R.H. (1987) Chem. Rev. 87, 1401-1449. Holm, R.H. (1990) Coord. Chem. Rev. 100, 183-221. Schultz, B.E., Gheller, S.F., Muetterties, M.C., Scott, M.J. and Holm, R.H. (1993) J. Am. Chem. Soc. 115, 2714-2722. Schultz, B.E. and Holm, R.H. (1993) Inorg. Chem. 32, 4244-4248. Caradonna, J.P., Reddy, P.R. and Holm, R.H. (1988) J. Am. Chem. Soc. 110, 2139-2144. Roberts, S.A., Young, C.G., Cleland, W.E., Ortega, R.B. and Enemark, J. H. (1988) Inorg. Chem. 27, 3044-3051. Xiao, Z., Young, C.G., Enemark, J.H. and Wedd, A.G. (1992) J. Am. Chem. Soc. 114, 9194-9195. Schultz, B.E., Hille, R. and Holm, R.H. (1995) J. Am. Chem. Soc. 117, 827-828. Bray, R.C., Gutteridge, S., Lamy, M.T. and Wilkinson, T. (1983) Biochem. J. 211,227-236. Howell, L.G. and Fridovich, I. (1968) J. Biol. Chem. 243, 59415947. Cohen, H.J. and Fridovich, I. (1971) J. Biol. Chem. 246, 359-6. Rajagopalan, K.V. (1980) in Molybdenum and Molybdenum Containing Enzymes (Coughlan, M, ed.), pp. 243-272, Pergamon Press, New York Kessler, D.L. and Rajagopalan, K.V. (1974) Biochim. Biophys. Acta 370, 389-398. Lamy, M.T, Gutteridge, S. and Bray, R.C. (1980) Biochem. J. 185, 397 -405. Bray, R.C., Gutteridge, S., Lamy, M.T. and Wilkinson, T. (1982) Biochem. J. 201,241-243. George, G.N. (1985) J. Mag. Res. 64, 384-394. George, G.N., Prince, R.C., Kipke, C.A., Sunde, R.A. and Enemark, J.H. (1988) Biochem. J. 256, 307-309. Gutteridge, S., Lamy, M.T. and Bray, R.C. (1980) Biochem. J. 191, 285-288. Cramer, S.P., Johnson, J.L. and Rajogopalan, K.V. (1979) Biochem. Biophys. Res. Commun. 91,434-439. Guthrie, J.P. (1978) Can. J. Chem. 56, 2342-2354. Kipke, C.A., Enemark, J.H. and Sunde, R.A. (1989) Arch. Biochem. Biophys. 270, 383-390. Kessler, D.L. and Rajagopalan, K.V. (1972) J. Biol. Chem. 247, 6566-6573. Massey, V. (1959) Biochim. Biophys. Acta 34, 255-256. Sullivan, Jr., E.P., Hazzard, J.T., Tollin, G. and Enemark, J.H. (1992) J. Am. Chem. Soc. 114, 9662-9663. Rohlfs, R.J. and Hille, R. (1994) J. Biol. Chem. 269, 30869-30879. Sullivan, E.P., Jr., Hazzard, J.T., Tollin, G. and Enemark, J.H. (1993) Biochemistry 32, 12465-12470.
ELSEVIER
et Biophysica A~ta Biochimica et Biophysica Acta 1253 (1995) 133-135
Rapid Report
The reaction of chicken liver sulfite oxidase with dimethylsulfite Michael S. Brody, Russ Hille * Department of Medical Biochemistry, The Ohio State University, 1645 Neil Avenue, 333 Hamilton Hall, Columbus, OH 43210-1218, USA Received 18 August 1995; accepted 28 August 1995
Abstract
We have undertaken a steady-state and rapid kinetic study of the reaction of enzyme with sulfite and dimethylsulfite. Methylation of sulfite results in a sign:ificant increase in K m and Kd for the substrate in the course of steady-state and rapid reaction kinetics, respectively, but kcat and the limiting rate constant for enzyme reduction (kred) are essentially unchanged. This indicates that while substrate oxyanion groups are effective in stabilizing the Eox. S complex, the breakdown of this complex proceeds at the same rate even in their absence. The critical element of the substrate required for reactivity is a suitable lone-pair available to undertake nucleophilic attack on a Mo=O group of the active site. Keywords: Oxo molybdenum enzyme; Enzyme reaction mechanism; Metalloenzyme
Sulfite oxidase (EC 1.8.3.1) catalyzes the oxidation of sulfite to sulfate with subsequent reduction of two equivalents of cytochrome c in the process; the oxygen atom incorporated into substrate is ultimately derived from water. The enzyme is one of the better understood members of the oxo molybdenum class of enzymes possessing a Mo w 0 2 unit in the active site [1], and the fundamental chemistry catalyzed by these enzymes has been investigated using several different model systems possessing a MoVIO2 unit [2-9]. These model compounds possess the ability to catalyze oxygen atom transfer between an oxo donor and acceptor by cycling between LnMoVlO2 and LnMoWO species, and the suitability of a given donor:acceptor pair for effective turnover has been adequately justified on thermodynamic grounds [4]. Phosphines have proven to be particularly useful as oxo acceptor species, reacting with LnMoWO2 to give LnMolVO and the corresponding phosphine oxide. Based on the negative activation e~Ltropies observed for reactions of the type MoWO2 + :PR 3 --~ MolVO + OPR 3, it has been concluded that the reaction proceeds via an associative transi-
* Corresponding author. Fax: + 1 (614) 292 4118. 0167-4838/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 5 ) 0 0 1 9 4 - 8
tion state formed by nucleophilic attack of the phosphine lone pair on one of the M o = O groups [7]:
C. OC-':PR
[ •. LMolV=O + O=-PR3
At least one oxo molybdenum enzyme, dimethylsulfoxide reductase from Rhodobacter sphaeroides, is able to use the water-soluble phosphine 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane as a reducing substrate in a reaction directly analogous to that in which the model compounds participate [10]. These observations emphasize the strong analogy between the chemistry of the model compounds and enzymes. The reaction of sulfite oxidase with sulfite might reasonably be expected to proceed in an analogous fashion to that proposed for the phosphine in the model compound chemistry, with the sulfur lone pair of sulfite undergoing nucleophilic attack on a M o = O group. Alternatively, it has been suggested that the catalytic sequence of sulfite oxidase begins with direct coordination of sulfite to the molybdenum via one of the oxyanion groups [11]. This view is consistent with the known ability of a variety of anions to inhibit sulfite oxidase [12-15], and to perturb the
134
M.S. Brody, R. Hille / Biochimica et Biophysica Acta 1253 (1995) 133-135
Mo v EPR signal of partially reduced enzyme [16-21]. In an effort to gain further insight into the reaction mechanism of sulfite oxidase, we have undertaken a steady-state and rapid kinetic study of the reaction of enzyme with sulfite and dimethylsulfite (the rate of hydrolysis of dimethylsulfite in water is 10 -5 s -1 [22]). We find that while methylation of sulfite results in a significant increase in K m and K 0 for the substrate in the course of steady-state and rapid reaction kinetics, respectively, kcat and the limiting rate constant for enzyme reduction (kr¢0) are unchanged. These results indicate that while substrate oxyanion groups are effective in stabilizing the Eox • S complex, the breakdown of this complex proceeds at the same rate even in their absence. We conclude that the critical element of the substrate required for reactivity is a suitable lone-pair available to undertake nucleophilic attack on a M o : O group of the active site. Steady state analysis following the catalytic velocity of sulfite oxidase J as a function of the sulfite and dimethylsulfite concentration under conditions of excess cytochrome c gave typical hyperbolic plots. A kca t of 79 S- 1 and a K m of 28 ~ M are found for sulfite, the K m in good agreement with published results [26]. The corresponding values obtained using dimethylsulfite are 89 s - ' for kca t and 6.3 mM for Kin. The close agreement of kcat for sulfite and dimethylsulfite suggests that breakdown of the Eox. S complex is not affected by substrate methylation; instead, the principal effect of methylation is on Km. The present results give a value for k c a t / K m of 2.8 X 10 6 M -1 s - I with sulfite and 1.4. 10 4 M -1 s - ! with dimethylsulrite. In order to gain greater insight into the reaction of sulfite and dimethylsulfite with sulfite oxidase, the reaction of enzyme with the two substrates has been examined by stopped-flow spectrophotometry, using the absorbance
i Sulfite oxidase was purified by a previously described method [23], with some modifications, involving homogenization of fresh chicken livers in liquid nitrogen, followed by centrifugation, ammonium sulfate fractionation and sequential chromatography on DEAE-Sepharose, Sephacryl-S-200, and a phenyl-Sepharose column. Damage to the molybdenum center was reduced by avoiding an acetone extraction in this procedure. The purified enzyme possessed a heme to protein absorbance ratio (A 414//A 280) o f > 0.5 and specific activity of 2.1- 103 ~ m o l / s - mg. Unless otherwise stated, all experiments were performed at 250(2 in 20 mM Tris adjusted to pH 8.0 with acetic acid, containing 2 mM Na2MoO 4 and 0.1 mM EDTA. The steady state experiments were performed aerobically using a saturating concentration of cytochrome c, 21 /xM (10-fold greater than K m) [24] and varying the concentration of either sulfite or dimethylsulfite. Rates were determined by following the reduction of cytochrome c at 550 nm [25], using an extinction change of 19 630 M - i c m - l . This emperically determined number was used because of the lower spectral resolution of the diode array spectrophotometer used in the present work. Cytochrome c [horse heart, (96% pure)] was obtained from Sigma. Dimethylsulfite ( > 99% pure) was obtained from Aldrich. All other chemicals were of reagent grade and used without additional purification.
0.10
A 0.08
0.06 ,.o O 0.04
0.02
0.00 0.0
!
I
0.1
0.2
i
I
i
0.3
0.4
1/[Sulfite] (uM) 0.10
0.08
0,06 O m
0.04
0.02
O.OG
.
0.0
.
.
.
~
0.5
.
.
.
.
J
.
1.0
.
.
.
1.5
1/[dimethylsulfite] (raM) Fig. 1. Double reciprocal plots for the reaction of sulfite oxidase with sulfite (Panel A) and dimethylsulfite (Panel B). Sulfite oxidase (0.4/zM in the reaction with sulfite, 0.5 #,M in the reaction with dimethylsulfite) was reacted with varying concentrations of substrate in a stopped-fiow apparatus, with the reaction monitored spectrophotometrically at 426 nm. The kinetic constants kred and K d, obtained from the reciprocal of the y-axis intercept and negative of the reciprocal of the x-axis intercept, respectively, are 194 s - 1 and 3 3 / z M for sulfite, and 170 s - i and l I mM for dimethylsulfite. Reaction conditions were 0.02 M tris-acetate buffer ( p n 8.0), 25°C.
change of the enzyme heme as a spectroscopic probe. 2 The reduction of oxidized sulfite oxidase at pH 8.0 by sulfite or dimethylsulfite under anaerobic conditions exhibits well-behaved single-exponential kinetics at all substrate concentrations which are independent of the wavelength monitored. Double reciprocal plots of the observed rate constant vs. substrate concentration for the reaction of enzyme with sulfite (Fig. 1A) and dimethylsulfite (Fig. 1B) are linear, giving a kred of 194 s -1 and a K d of 33
2 Rapid kinetics experiments were performed anaerobically with fully oxidized enzyme using methods described previously [27]. In the present analysis, it is assumed that intramolecular electron transfer from the molybdenum center to the heme of sulfite oxidase is as rapid as was seen in a related study by Sullivan et al. [28], and rate-limited by the chemistry taking place at the molybdenum center.
M.S. Brody, R. Hille / Biochimica et Biophysica Acta 1253 (1995) 133-135
/zM for sulfite; the corresponding values for dimethylsulrite are 170 s-1 and 11 mM. A comparison of the kre d values for the two substrates indicates that this rate constant, which represents the breakdown of the Eox • S complex to Ered + P, is essentially unaffected by methylation of the substrate, consistent with the steady-state results. Methylation of substrate results in a 300-fold increase in K d, indicating a sub:~tantial reduction in the affinity of enzyme for substrate. Overall, these results indicate that while the oxyanion :groups of sulfite are important for substrate binding, perhaps by direct coordination to molybdenum, they are not required for catalysis. A comparison of i~ca t and kre d for sulfite (79 s-1 and 194 s -1, respectively) indicates that the later value is substantially larger. This implies that the reaction of oxidized enzyme with stllfite (or dimethylsulfite) is not ratelimiting in overall turnover for sulfite oxidase and that reaction of reduced enzyme with cytochrome c in the oxidative half of the catalytic cycle must contribute significantly in limiting turnover rate. The oxo molybdenum enzymes may be subdivided into Mo w 02- and Mo v~OS-containing categories [ 1]. The latter group of enzymes, with the exception of molybdenum-containing CO dehydrogenase from species such as Pseudomonas carboxydo~orans, catalyze hydroxylation reactions at carbon centers that necessarily entail cleavage of a C - H bond. The sulfido group is essential for this reaction, as its replacement with a second M o : O group (by reaction with cyanide) renders these enzymes inactive, and it presumably plays a direct role in the cleavage of the C - H bond. The MoVIO2 group of enzymes, including sulfite oxidase, catalyzes a possibly more facile oxygen atom transfer into or out of heteroatom centers (nitrogen, sulfur or arsenic). These re~.ctions invariably involve the reaction of oxidized enzyme with an oxo acceptor possessing a lone electron pair (e.g., sulfite oxidase) or reduced enzyme with an oxo donor to give a product that possesses a lone pair (e.g., nitrate reductase or DMSO reductase). In the case of sulfite oxidase, the present results indicate that it is the availability of a lone pair capable of accepting an oxo group that is the principal element determining suitability as a substrate for tJae enzyme; methylation of the two oxyanion groups reduces the affinity of substrate for the active site, but once the Michaelis complex is formed (at sufficiently high substrate concentration) it breaks down with the same rate ctmstant as does sulfite. In the absence of steric effects due 1:o methylation, the large difference in K d observed for sulfite and dimethylsulfite indicates that substrate oxyanions contribute 3.4 kcal/mol toward the stabilization of the l~.ox • S complex, consistent with direct coordination to the molybdenum (although participation in an electrostatic or hydrogen bonding interactions with other functional groups of the active site cannot be excluded). Dimethylsulfite does exhibits saturation kinetics,
135
however, implying that a pre-equilibrium (Michaelis) complex is formed with this substrate and that the interactions between substrate and active site other than those involving oxyanions must also contribute to stabilization of the complex.
Acknowledgements This work was supported by National Institute of Health grant number AR38917.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
[15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28]
Hille, R. (1994) Biochim. Biophys. Acta 1184, 143-169. Holm, R.H. and Berg, J.M. (1986) Acc. Chem. Res. 19, 363-370. Holm, R.H. (1987) Chem. Rev. 87, 1401-1449. Holm, R.H. (1990) Coord. Chem. Rev. 100, 183-221. Schultz, B.E., Gheller, S.F., Muetterties, M.C., Scott, M.J. and Holm, R.H. (1993) J. Am. Chem. Soc. 115, 2714-2722. Schultz, B.E. and Holm, R.H. (1993) Inorg. Chem. 32, 4244-4248. Caradonna, J.P., Reddy, P.R. and Holm, R.H. (1988) J. Am. Chem. Soc. 110, 2139-2144. Roberts, S.A., Young, C.G., Cleland, W.E., Ortega, R.B. and Enemark, J. H. (1988) Inorg. Chem. 27, 3044-3051. Xiao, Z., Young, C.G., Enemark, J.H. and Wedd, A.G. (1992) J. Am. Chem. Soc. 114, 9194-9195. Schultz, B.E., Hille, R. and Holm, R.H. (1995) J. Am. Chem. Soc. 117, 827-828. Bray, R.C., Gutteridge, S., Lamy, M.T. and Wilkinson, T. (1983) Biochem. J. 211,227-236. Howell, L.G. and Fridovich, I. (1968) J. Biol. Chem. 243, 59415947. Cohen, H.J. and Fridovich, I. (1971) J. Biol. Chem. 246, 359-6. Rajagopalan, K.V. (1980) in Molybdenum and Molybdenum Containing Enzymes (Coughlan, M, ed.), pp. 243-272, Pergamon Press, New York Kessler, D.L. and Rajagopalan, K.V. (1974) Biochim. Biophys. Acta 370, 389-398. Lamy, M.T, Gutteridge, S. and Bray, R.C. (1980) Biochem. J. 185, 397 -405. Bray, R.C., Gutteridge, S., Lamy, M.T. and Wilkinson, T. (1982) Biochem. J. 201,241-243. George, G.N. (1985) J. Mag. Res. 64, 384-394. George, G.N., Prince, R.C., Kipke, C.A., Sunde, R.A. and Enemark, J.H. (1988) Biochem. J. 256, 307-309. Gutteridge, S., Lamy, M.T. and Bray, R.C. (1980) Biochem. J. 191, 285-288. Cramer, S.P., Johnson, J.L. and Rajogopalan, K.V. (1979) Biochem. Biophys. Res. Commun. 91,434-439. Guthrie, J.P. (1978) Can. J. Chem. 56, 2342-2354. Kipke, C.A., Enemark, J.H. and Sunde, R.A. (1989) Arch. Biochem. Biophys. 270, 383-390. Kessler, D.L. and Rajagopalan, K.V. (1972) J. Biol. Chem. 247, 6566-6573. Massey, V. (1959) Biochim. Biophys. Acta 34, 255-256. Sullivan, Jr., E.P., Hazzard, J.T., Tollin, G. and Enemark, J.H. (1992) J. Am. Chem. Soc. 114, 9662-9663. Rohlfs, R.J. and Hille, R. (1994) J. Biol. Chem. 269, 30869-30879. Sullivan, E.P., Jr., Hazzard, J.T., Tollin, G. and Enemark, J.H. (1993) Biochemistry 32, 12465-12470.