Manganese stimulation and stereospecificity of the Dopa (3,4-dihydroxyphenylalanine)/tyrosine-sulfating activity of human monoamine-form phenol sulfotransferase. Kinetic studies of the mechanism using wild-type and mutant enzymes.

Kinetic studies were performed to dissect the mechanism underlying the remarkable Mn(2+) stimulation of the Dopa/tyrosine-sulfating activity of the human monoamine (M)-form phenol sulfotransferase (PST). The activities and the stimulation by Mn(2+) are highly stereospecific for the d-form enantiomers of tyrosine and Dopa. Analysis of the kinetic results strongly suggests that tyrosine-Mn(2+) and tyrosine-Mn(2+)-tyrosine complexes are obligatory substrates, whereas Dopa-Mn(2+) complexes may be better substrates than Dopa alone. This activation of the Dopa/tyrosine-sulfating activity of M-form PST by Mn(2+) via complex formation between Mn(2+) and the substrate is the first reported case of a regulatory mechanism in this important class of enzymes. Our previous studies using point-mutated M-form PSTs established that the Mn(2+) (in the substrate-Mn(2+) complex) exerts its stimulatory effect by binding predominantly to the Asp-86 residue at the active site. We present here further studies using dopamine as substrate to bolster this conclusion. The possible physiological implications of this rather unusual specificity for the d-amino acid and its derivatives and the stimulation by Mn(2+) are discussed in the context of protective and detoxification mechanisms that may operate in neurodegenerative processes in the brain. The Mn(2+) stimulation of the activity of M-form PST toward d-enantiomers of Dopa/tyrosine may have implications for other substrates (including chiral drugs) and for the other cytosolic sulfotransferases that are involved in the regulation of endogenous metabolites as well as in detoxification.

Sulfotransferases (STs) 1 are enzymes ubiquitous in both plants and animals that catalyze the sulfation of a variety of compounds containing hydroxyl or amino groups using 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS) as the sulfonyl group donor (1)(2)(3). Although the membrane-bound STs use proteins, glycolipids, and other macromolecules as substrates, the cytosolic STs sulfate smaller molecules and are part of the Phase II detoxification pathway for the biotransformation/excretion of drugs and xenobiotics. This serves to both detoxify dietary, therapeutic, and environmental xenobiotics as well as regulate the levels and activities of endogenous molecules such as thyroid and steroid hormones, catecholamine hormones/neurotransmitters, and bile acids (4,5). Except during the early stage of development, cytosolic STs in general have been shown to be constitutive enzymes with little known about the regulation of their enzymatic activities (6,7). In the past several years, however, studies performed in our laboratory reveal that Mn 2ϩ exerts a stimulatory effect on sulfation of some substrates by the human monoamine (M)-form phenol sulfotransferase (PST) (8,9).
The human M-form PST is the only sulfotransferase that sulfates the catecholamines, in particular the neurotransmitter dopamine, with high activity (4). This enzyme is found in the upper gastrointestinal tract, brain, platelet, and lung (10). In the gastrointestinal tract it may detoxify potentially lethal dietary catecholamines. In the brain it may play a role in regulating the levels of dopamine. We had previously demonstrated that besides its activity toward catecholamines, M-form PST could uniquely sulfate the free amino acid form of tyrosine and 3,4-dihydroxyphenylalanine (Dopa) (8,9,11). Interestingly, it showed higher activities toward the D-enantiomers (as compared with the L-enantiomers) of these compounds and a remarkable stimulation (by more than 100-fold) of the activities by sub-millimolar and millimolar levels of Mn 2ϩ , especially with the D-enantiomers. Mn 2ϩ also stimulates the activity with dopamine, although only 2-3-fold (9). Mn 2ϩ is known to be present at higher levels in human neuronal tissue (12) and is sequestered intracellularly in mitochondria (13). Oxidative stress or damage, which has been implicated in neuronal apoptosis that occurs in neurodegenerative diseases, generally results in mitochondrial dysfunction (14). The consequent release of Mn 2ϩ into the cytosol may activate the M-form PST and, in particular, its Dopa/tyrosine-sulfating activity. It has also been observed that D-amino acids accumulate in aging tissues, especially if the levels of D-amino acid oxidases are low (15). Attempts have been made to link the amount of specific D-amino acids to oxidative damage and to neurodegenerative disorders such as Alzheimer's and Parkinson's diseases (16,17). A clear picture is yet to emerge. However, the removal of D-amino acids by sulfation may be viewed as a detoxification process. From a different perspective, the sulfation of D-tyrosine could also serve as a useful model in the study of the stereospecific action of the PST enzymes on chiral drugs (18 -21). It should also be emphasized that the dramatic stimulation by Mn 2ϩ of the sulfation of these substrates may be part of a more general mechanism to increase the promiscuity of M-form PST toward unusual or xenobiotic substrates in the presence of a molecular trigger such as increased Mn 2ϩ concentrations.
In this paper, we report kinetic studies on the sulfation of dopamine and the D-and L-enantiomers of tyrosine and Dopa by the wild-type M-form PST and its Asp-86 point mutant and the stimulation by Mn 2ϩ . M-form PST is known to exist as a homodimer in its native state (22), and the reported x-ray structure of the protein (23) revealed that residues 84 -92 of one subunit form a "mobile" loop that may intercalate into the active site of the other subunit. It was suggested that the presence of this mobile loop might hinder the proper positioning of some substrates (23). Our previous studies (11) have established the importance of two regions in the sequence of M-form PST, designated Region I (spanning residues 84 -89) and Region II (residues 143-148), to its dopamine-sulfating activity as well as its Dopa/tyrosine-sulfating activity and the Mn 2ϩ stimulation. These are the regions that vary between M-form PST and the P-form PST (that does not possess Mn 2ϩstimulated Dopa/tyrosine-sulfating activity), which otherwise are more than 93% identical in their amino acid sequences (11). That the Region I is part of the above-mentioned mobile loop intercalating into the active site allows for the formulation of an attractive model to explain our kinetic results. Our previous studies with point mutants in Regions I and II have also underlined the importance of residues Asp-86 and Glu-89 in Region I and of residue Glu-146 in Region II in the dopaminesulfating activity of M-form PST (24). Further studies with such point mutants and two deletional mutants (lacking residues 84 -90 and 84 -86, respectively, of the purported loop intercalating into the active site in the wild-type M-form PST) have revealed that both the loop as a whole (rather than the residues comprising it) as well as residue Glu-146 in Region II are important to the stereospecificity of M-form PST for the D-enantiomers of Dopa and tyrosine. Residue Asp-86 in Region I, on the other hand, is the one most important to the Mn 2ϩ dependence of this activity. 2 We also present in this paper studies with the D86A point mutant to further dissect the structural basis for these activities and their activation by Mn 2ϩ .
Bacterial Expression and Purification of the Recombinant Human Wild-type and D86A Point-mutated M-form PSTs-Competent E. coli BL21 cells transformed with pGEX-2TK vector harboring the wild-type or D86A point-mutated M-form PST cDNA were grown to A 600 nm ϭ 0.8 in 1 liter of LB medium supplemented with 50 g/ml ampicillin. After induction with 0.1 mM isopropyl ␤-D-thiogalactopyranoside overnight at room temperature, the cells were collected by centrifugation and homogenized in 25 ml of an ice-cold lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, and 10% glycerol using an Amicon French press. Ten l of a 10 mg/ml aprotinin solution was added to the homogenate, which was then centrifuged at 10,000 ϫ g for 15 min at 4°C. The supernatant collected was fractionated by equilibrating with 1.5 ml of glutathione-Sepharose for 20 min at 4°C, and the supernatant and the washings with the lysis buffer were discarded. The bound fusion protein was treated with 3 ml of a thrombin digestion buffer (containing 5 units/ml bovine thrombin, 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2.5 mM CaCl 2 , and 10% glycerol). After a 30-min incubation at room temperature, the preparation was centrifuged. The recombinant enzyme present in the supernatant was collected and analyzed by SDS-PAGE, and the protein concentration was determined and used in the enzymatic assays. Ten mg/ml of bovine serum albumin was added as a stabilizing agent to this preparation, that was then stored in small aliquots at Ϫ70°C until use.
Enzymatic Assay-The sulfotransferase assays were performed using [ 35  The enzyme dilutions were prepared in 50 mM TAPS, pH 8.25, or 50 mM HEPES, pH 7.0, containing 10% glycerol. The MnCl 2 and enzyme solutions were added last to the reaction mixture, which was immediately incubated for 3 min at 37°C. The reaction was stopped by the addition of 5 l of 2.5 M acetic acid, vortexed, and centrifuged to clear any precipitates (26). The amount of enzyme chosen was such as to ensure that there was not more than 5% reaction so that the reaction was linear with time and amount of enzyme. The final reaction mixture was subjected to the analysis of i 35 S-sulfated product by spotting a 3-l aliquot onto the cellulose TLC plate, which was then subjected to ascending TLC using a solvent system containing n-butanol, isopropanol, 88% formic acid, H 2 O in a 3:1:1:1 ratio by volume. In the case of Dopa and dopamine, where the sulfated product migrated too close to unused [ 35 S]PAPS for efficient separation or whenever the background was too strong, a two-dimensional separation was performed on the samples spotted on the TLC plate by first running a high voltage (1000 volts) thin-layer electrophoresis in the first dimension followed by the above-mentioned ascending TLC in the second dimension (27). Afterward, the plates were air-dried and autoradiographed. The radioactive spots on the TLC plates due to 35 S-sulfated products were cut out and eluted by shaking in 0.5 ml of H 2 O in glass vials. Four ml of scintillation fluid was then added to each vial and thoroughly mixed, and the radioactivity was counted using a liquid scintillation counter. The counts obtained were used to calculate the specific activity of the enzyme under the particular reaction conditions in units of nmol of sulfated product formed/min/mg of enzyme. Assays were performed in triplicate, and a control without enzyme was installed to correct for any background counts.
Miscellaneous Methods-[ 35 S]PAPS (carrier-free) was synthesized from ATP and carrier-free [ 35 S]sulfate using the human bifunctional ATP sulfurylase/adenosine 5Ј-phosphosulfate kinase, and its purity was determined as described previously (28). The [ 35 S]PAPS synthesized was then adjusted to the required concentration and specific activity by the addition of cold PAPS. The concentration of PAPS was confirmed by measuring its absorbance at 260 nm (29). SDS-PAGE was performed on 12% polyacrylamide gels using the method of Laemmli (30). Protein determination was based on the method of Bradford with bovine serum albumin as the standard (31).

RESULTS
Our previous studies demonstrate that M-form PST, besides sulfating dopamine, could also sulfate tyrosine and Dopa (8,9,11). Some interesting features of these latter activities were (i) that these activities can be dramatically stimulated by Mn 2ϩ and (ii) that the enzyme shows higher activities toward the D-rather than the L-enantiomers of tyrosine and Dopa. By taking a kinetic approach along with studies using mutated M-form PSTs, the current study was aimed at investigating the underlying mechanism for the Mn 2ϩ stimulation and stereospecificity.

Kinetics of Sulfation of Dopamine by M-form PST in the Presence of Varying Concentrations of Mn 2ϩ -
We first studied the modest stimulation by Mn 2ϩ of the sulfation of dopamine by M-form PST (8,9). Dopamine is believed to be the physiological substrate for M-form PST. Because it has no optical isomers and contains no carboxyl group as in Dopa and tyrosine, it was interesting to investigate the extent and mechanism of the stimulation by Mn 2ϩ of dopamine sulfation by M-form PST. The kinetics of the sulfation of dopamine by M-form PST was studied using varying concentrations (ranging from 0.5 M to 50 M) of dopamine, in the presence of different concentrations (0, 0.1, 0.5, 1.0, 2.5, 5.0 mM) of Mn 2ϩ or in the presence of 1 mM EDTA (as a control). It had been demonstrated that the dopamine-sulfating activity of M-form PST was maximal at pH 7.0 (22). HEPES buffer at pH 7.0 was therefore used in this study along with a saturating PAPS concentration of 15 M. Fig. 1 shows the plots of the velocity (v) versus substrate (dopamine) concentration ([S]) in the presence of different concentrations of Mn 2ϩ . It is clear from these plots that regardless of the Mn 2ϩ concentration, V max was reached with 10 -20 M dopamine concentrations. Mn 2ϩ appeared to increase the V max while changing K m for dopamine only slightly. In the presence of 1 mM EDTA, the V max was 370 nmol/min/mg, and the K m was 2.4 M, whereas with 5 mM Mn 2ϩ , the V max was 1000 nmol/min/mg, and the K m was 4.5 M. The kinetics appeared to be of the Michaelis-Menten type.
Effects of Mn 2ϩ on the Sulfation of Dopamine by the D86A Point Mutant of M-form PST-Our recent studies show that, although Mn 2ϩ has a remarkable stimulatory effect on the sulfation, especially of the D-enantiomers of Dopa and tyrosine (Refs. 8 and 10) by M-form PST, the D86A point mutant of this enzyme showed none or just a marginal Mn 2ϩ stimulation of sulfation of these substrates. 2 This was interpreted to imply that the stimulatory effect of Mn 2ϩ is exerted predominantly via its binding to the negatively charged residue Asp-86, which from the x-ray diffraction studies is believed to be part of the mobile loop intercalating into the active site of M-form PST (23). To find out whether residue Asp-86 also mediates the modest Mn 2ϩ stimulation of the sulfation of dopamine, the sulfation of dopamine (at 20 M) by the wild-type M-form PST and the D86A point mutant in the absence or presence of 5 mM Mn 2ϩ was studied. The results tabulated in Table I showed clearly that not only were the activity levels of the D86A point mutant lower, but the stimulatory effect of Mn 2ϩ seemed to have been lost.

Kinetics of Sulfation of D-Tyrosine by M-form PST in the Presence of Varying Concentrations of Mn 2ϩ -
The kinetics of the sulfation of D-tyrosine by M-form PST was studied using varying concentrations (ranging from 0.1 to 10 mM) of D-tyrosine in the presence of different concentrations (0, 0.1, 0.5, 1.0, 2.5, 5.0, and 10 mM) of Mn 2ϩ or in the presence of 1 mM EDTA (as a control). We had previously demonstrated that this activity was maximal between pH 8.0 and 9.0 (8). TAPS buffer at pH 8.25 was therefore used in this study. It was first established that the PAPS concentration used (15 M) was saturating, since there was no appreciable increase in the velocity of the reaction even when a 10-fold higher concentration of PAPS was used at several different D-tyrosine and Mn 2ϩ concentrations (such studies were also repeated with the other substrates subsequently used). Fig. 2 shows the plots of the velocity (v) versus total substrate (D-tyrosine) concentration ([S] t ) in the presence of different concentrations of Mn 2ϩ (the S t profiles). It is evident from these plots that at 10 mM D-tyrosine, although saturation with substrate was reached in the presence of 1, 2.5, or 5 mM Mn 2ϩ , the curves still reached maximal velocity in the presence of lower concentrations of Mn 2ϩ (0, 0.1, and 0.5 mM). However, D-tyrosine has a limited solubility in water, and it was difficult to prepare stable solutions containing greater than 10 mM D-tyrosine under the assay conditions to show the extended curves at lower concentrations of Mn 2ϩ . On the other hand, concentrations of Mn 2ϩ higher than 5 mM resulted in precipitation and, consequently, inconsistent results at higher concentrations of D-tyrosine. Therefore, with 10 mM Mn 2ϩ , only the data obtained at D-tyrosine concentrations of 5 mM or less were analyzed. This is clear from the plots of velocity (v) versus total Mn 2ϩ ([A] t ) concentration in the presence of different concentrations of substrate D-tyrosine shown in Fig. 3 (the A t profiles).  The plots shown in Fig. 2 demonstrated clearly that Mn 2ϩ had a remarkable stimulatory effect on the sulfation of D-tyrosine by M-form PST. Fig. 2 also shows that at 5 mM Mn 2ϩ (and at 10 mM Mn 2ϩ ; data not shown), some inhibition started occurring in the presence of higher concentrations of D-tyrosine.

Stereospecificity of M-form PST; Lower Activity, Affinity, and Mn 2ϩ Stimulation with L-Tyrosine Relative to D-Tyrosine as
Substrate-The kinetics of sulfation of L-tyrosine at various concentrations ranging from 0.5 to 10 mM by M-form PST in the presence of either 1 mM EDTA or 0 or 5 mM Mn 2ϩ was studied. Fig. 4 shows the corresponding velocity versus [S] plots. From these plots, it is clear that saturation with substrate was not reached even at 10 mM L-tyrosine in the presence of 5 mM Mn 2ϩ . As in the case of D-tyrosine, solubility and precipitation problems made it unfeasible to extend the studies to higher concentrations of L-tyrosine or Mn 2ϩ . However, it is clear from Fig. 4 that the affinity of M-form PST for L-tyrosine is very much lower than for D-tyrosine. With D-tyrosine as substrate, saturation was reached at ϳ7 mM with 2.5 mM Mn 2ϩ and at 5 mM with 5 mM Mn 2ϩ , whereas with L-tyrosine as substrate, saturation was not reached even at 10 mM with 5 mM Mn 2ϩ . The specific activities at different substrate and Mn 2ϩ concentrations were also found to be much lower with L-tyrosine than with D-tyrosine as substrate (e.g. 750 nmol/min/mg for 5 mM D-tyrosine at 5 mM Mn 2ϩ versus 6 nmol/min/mg for 5 mM L-tyrosine at 5 mM Mn 2ϩ ). Moreover, the stimulatory effect of Mn 2ϩ on the sulfation of L-tyrosine was much less dramatic than with D-tyrosine as substrate.
Kinetics It is interesting to point out that the affinity of M-form PST for D-Dopa appeared to be much higher than for D-tyrosine. With D-tyrosine, saturation was reached at about 7 mM with 2.5 mM Mn 2ϩ and at 5 mM with 5 mM Mn 2ϩ , whereas with D-Dopa, saturation was reached at 0.5 mM with 2.5 mM Mn 2ϩ . The specific activities at different substrate and Mn 2ϩ concentrations were also much higher with D-Dopa than with D-tyrosine as substrate (e.g. 750 nmol/min/mg for 5 mM D-tyrosine at 5 mM Mn 2ϩ versus 1200 nmol/min/mg for 0.5 mM D-Dopa at 2.5 mM Mn 2ϩ ). However, the stimulatory effect of Mn 2ϩ on the sulfation of D-Dopa is less dramatic than with D-tyrosine as substrate.

Kinetics of Sulfation of L-Dopa by M-form PST; Lower Affinity and Mn 2ϩ Stimulation Compared with the Sulfation of D-Dopa-
The kinetics of sulfation of L-Dopa at various concentrations ranging from 25 to 2500 M by M-form PST in the presence of 0 mM Mn 2ϩ or 2.5 mM Mn 2ϩ was studied. Fig. 6 shows the corresponding v versus [S] plots. With or without Mn 2ϩ it appeared that there was no saturation with substrate even at 2500 M L-Dopa, which approached the solubility limit for L-Dopa under the assay conditions. However, as in the case of the tyrosine enantiomers, the affinity of M-form PST for L-Dopa seemed to be very much lower than for D-Dopa, and the stimulatory effect of Mn 2ϩ on the sulfation of L-Dopa was much less dramatic than for D-Dopa.

Model to Explain the Stimulatory Effect of Mn 2ϩ on Dopamine Sulfation by M-form PST-Our
recent studies have established that the stimulation by Mn 2ϩ of the Dopa/tyrosine-sulfating activities of M-form PST is primarily mediated by residue Asp-86 in variable Region I of the molecule (11). 2 This region has been shown by x-ray crystallography (23) to be part of a mobile loop formed by residues 84 -92 of one subunit that intercalates into the active site of the other subunit of this dimeric enzyme (22). Results from our current study on the sulfation of dopamine by the wild-type M-form PST (cf. Fig. 1) and its D86A point mutant (cf. Table I) indicated that the smaller stimulatory effect of Mn 2ϩ on the dopamine-sulfating activity of the wild-type enzyme was also mediated by the binding of Mn 2ϩ to the Asp-86 residue in the molecule. It is at present unclear how the binding of Mn 2ϩ to the Asp-86 residue at the active site stimulates the dopamine sulfation. Nevertheless, the stimulation was apparently due to an increase in V max , without significant effect on K m (cf. Fig. 1). The activity toward dopamine (with or without Mn 2ϩ ) basically followed Michaelis-Menten kinetics. This suggests that the binding of Mn 2ϩ to the Asp-86 residue may increase the catalytic efficiency of the enzyme while not affecting (or marginally hindering) the binding of dopamine at the active site. The kinetics does not suggest any involvement of a dopamine-Mn 2ϩ complex, as is to be expected because dopamine contains no negatively charged group to co-ordinate to the Mn 2ϩ . The dissociation constant for the binding of Mn 2ϩ to the enzyme at the Asp-86 residue appears to be in the mM range, based on the data presented. omers. Its activity toward these compounds was dramatically stimulated by Mn 2ϩ in the range of Mn 2ϩ concentrations up to 5 mM. The stimulation was found to be much more dramatic with the D-enantiomers and was evident at Mn 2ϩ levels as low as 0.1 mM. The kinetic plots in Figs. 2-6 indicated that the interaction of M-form PST with Dopa or tyrosine in the presence of Mn 2ϩ was co-operative in nature. This could be possibly be modeled for the behavior with tyrosine by a kinetic scheme where a tyrosine-Mn 2ϩ or a tyrosine-Mn 2ϩ -tyrosine complex is the real substrate (32,33). Because the activity with tyrosine alone was found to be negligible and that with tyrosine plus Mn 2ϩ considerably higher, the tyrosine-Mn 2ϩ and tyrosine-Mn 2ϩ -tyrosine complexes would likely be the obligatory substrates. In the case of Dopa, there was a considerable activity even without Mn 2ϩ , indicating that the Mn 2ϩ -Dopa complexes are not obligatory but rather better substrates compared with Dopa alone. Mn 2ϩ may bind to the negatively charged carboxyl group of one or two tyrosine/Dopa molecules to form the complexes. Our recent studies using point mutants suggest that a positively charged amino group of the tyrosine/Dopa-Mn 2ϩ complexes may interact mainly with the negatively charged Glu-146 residue at the active site of the enzyme. 2 Moreover, the positively charged Mn 2ϩ moiety of the complex may co-ordinate predominantly with the negatively charged Asp-86 residue of the mobile loop of the enzyme at the active site.
A search of the literature gave the log K for formation of the tyrosine-Mn 2ϩ complex (at 25°C and an ionic strength of 0.1; pH not specified) as 1.5 (molar concentrations used), whereas that for the formation of the tyrosine-Mn 2ϩ -tyrosine complex was 5.0 (34,35). We used these values as a first approximation to calculate the concentrations of the two complexes in the assay mixture with various total concentrations of Mn 2ϩ and tyrosine. The calculations were done using the equations for the two equilibria mentioned above together with the conditions [tyrosine] free ϭ [tyrosine] total Ϫ 2[tyrosine-Mn 2ϩ -tyrosine] Ϫ [tyrosine-Mn 2ϩ ] and [Mn 2ϩ ] free ϭ [Mn 2ϩ ] total Ϫ [tyrosine-Mn 2ϩ -tyrosine] Ϫ [tyrosine-Mn 2ϩ ] along with the requirement that real and positive numbers be involved. An iterative numerical procedure was used to solve these equations for the concentrations of the two complexes present when different amounts of Mn 2ϩ and tyrosine (total) are taken using a computer program written in visual basic. It is to be pointed out that the above equation showing the relationship between free and bound Mn 2ϩ concentrations does not take into consideration the possible binding of Mn 2ϩ to DTT, TAPS, and PAPS. An exhaustive literature search failed to find any association constants for the binding of these latter compounds to Mn 2ϩ . In the case of DTT, the pK a values for the two sulfhydryl groups had been determined to be 9.2 and 10.1, respectively (36). Because the assays with tyrosine as substrate in the present study were performed at pH 8.25, both the sulfhydryl groups of DTT would be predominantly uncharged and may not be expected to coordinate strongly to Mn 2ϩ under these conditions.
In Fig. 7, the velocity (in nmol of product formed/min/mg of protein) for the sulfation of D-tyrosine by M-form PST is plotted versus the total concentration of the D-tyrosine-Mn 2ϩ and D-tyrosine-Mn 2ϩ -D-tyrosine complexes (in moles/liter) in the assay mixture, calculated as indicated above. The data used are the same as presented in Fig. 2. It is clear that the data show Michaelis-Menten behavior in the interaction of the enzyme with these complexes. This is not the case if the concentration of only one of the two complexes is plotted on the x axis, indicating that the true substrates for the reaction include both the D-tyrosine-Mn 2ϩ as well as the D-tyrosine-Mn 2ϩ -D-tyrosine complexes. The K m and V max values of the enzyme for the two complexes, however, must differ because the residual charge on the Mn 2ϩ moiety in the D-tyrosine-Mn 2ϩ complex will be higher, and consequently, its interaction with the Asp-86 residue on the enzyme is expected to be stronger than in the case of the D-tyrosine-Mn 2ϩ -D-tyrosine complex. However, because we are dealing with a dynamic equilibrium, it is likely that, once the D-tyrosine-Mn 2ϩ -D-tyrosine complex enters and binds to the substrate pocket, the Asp-86 residue of the enzyme may bind to the Mn 2ϩ moiety, exchanging with the outer D-tyrosine residue and replacing it. Thus, this complex may also behave effectively like the D-tyrosine-Mn 2ϩ complex. The less than perfect fit to Michaelis-Menten behavior is to be expected considering the approximations made in using the values for the association constants for the formation of the complexes. The K m of the enzyme for these Mn 2ϩ -D-tyrosine complexes appears to be in the range of 0.75-0.85 mM, whereas the V max is in the range of 750 -850 nmol/min/mg. Our studies on dopamine sulfation by M-form PST as discussed above also indicated that Mn 2ϩ may bind to the enzyme, and it is likely that the inhibition at higher (mM) levels of Mn 2ϩ and tyrosine was due to their inhibition on the binding of the Mn 2ϩ -D-tyrosine complexes.
It is likely that similar calculations can be done to show that D-Dopa-Mn 2ϩ and D-Dopa-Mn 2ϩ -D-Dopa complexes are also responsible for the Mn 2ϩ stimulation of the D-Dopa-sulfating activity of M-form PST. However, no values of the association constants for the relevant complexes could be found in the literature, and also the analysis with D-Dopa will be compli- cated by the fact that Dopa has a substantial activity by itself. Similar scenarios can be postulated for the interaction with the L-isomers of tyrosine and Dopa.
The involvement of a Mn 2ϩ -PAPS complex as an obligatory or additional substrate appeared unlikely since saturation of PAPS was ensured in these experiments. Increasing the PAPS concentration (which was 15 M in the standard assay) 10-fold at several Mn 2ϩ concentrations did not result in any increase in reaction velocity. Moreover, our results clearly showed that the stimulatory effect of Mn 2ϩ on the sulfation activity of M-form PST is a function of a particular acceptor substrate. Another argument against a Mn 2ϩ -PAPS complex is the fact that no Mn 2ϩ requirement or stimulation has been reported for the activity of any of the other cytosolic STs (at least with their commonly used substrates), which all use PAPS as a co-substrate. Moreover, a comprehensive study performed in our laboratory on the effect of a variety of divalent metal ions on the activity of 10 known human cytosolic STs toward their commonly used substrates did not reveal any universal metal ion requirement or stimulation. 2 Stereospecificity of M-form PST and Its Relative Activity toward Dopa and Tyrosine-In this study, we found that in the presence of 5 mM Mn 2ϩ , the [S] 0.5 for D-tyrosine was around 1.9 mM, and the V max was around 750 nmol/min/mg, whereas for D-Dopa the [S] 0.5 was around 100 M, and the V max was around 1200 nmol/min/mg. Part of the difference may be due to the relative values of the dissociation constants for the two substrate-Mn 2ϩ complexes. Additionally, D-tyrosine can only be sulfated at the 4-OH group, whereas for D-Dopa, it has been demonstrated that sulfation occurs exclusively at the 3-OH group (37). The much greater affinity for D-Dopa relative to D-tyrosine can probably be explained by QSAR (quantitative structure activity relationship) analysis (23). The Mn 2ϩ -stimulated activity of M-form PST with, and its affinity for, the L-enantiomers of Dopa and tyrosine was far lower than with the D-enantiomers, which could probably also be explained by similar arguments and modeling studies (23). In our previous studies we had shown that the stereospecificity of M-form PST for the D-enantiomers of Dopa and tyrosine was mediated primarily by residue Glu-146 at the active site, which probably binds the positively charged amino groups of these substrates. 2 Point mutations of selected residues 84 -89 that are part of the putative loop at the active site did not significantly affect this stereoselective behavior. However, a residues-84 -90 deletional mutant of M-form PST showed no stereoselectivity in its sulfation activity toward tyrosine and Dopa. This suggested that although individual residues in the 84 -92 loop may not be critical for the stereoselectivity, the presence of the loop as such is essential, probably as an additional steric selector. The residues-84 -86 deletional mutant showed no activity toward any stereoisomer of either tyrosine or Dopa, possibly because the truncated loop may interfere with access of these substrates to the active site.
Physiological Relevance of the Stimulation of Sulfating Activity of M-form PST by Mn 2ϩ -In this study, the maximum stimulatory effect of Mn 2ϩ was observed at concentrations of around 5 mM. However, it was evident that significant stimulatory effects already occurred at levels as low as 0.1 mM. Mn 2ϩ is an important element biologically and has been shown to be essential to the activity of a number of enzymes in a variety of organisms (12,38,39). For example, it is central to the function of superoxide dismutase, an enzyme that protects against oxidative damage in tissues (12,8). The Mn 2ϩ concentrations in neuronal and brain tissue have been reported to be higher than in other tissues (12). Within the cell, Mn 2ϩ may be preferentially sequestered in mitochondria and endoplasmic reticulum (13). As stated previously, oxidative stress or damage, which has been implicated in neuronal apoptosis that occurs in neurodegenerative diseases, generally results in mitochondrial dysfunction (14) that may lead to the release of Mn 2ϩ into the cytosol. Elevated Mn 2ϩ concentration in the cytosol will stimulate M-form PST in its sulfating activity with dopamine and especially in its Dopa/tyrosine-sulfating activity. This may represent a detoxifying mechanism as discussed later. One such neurodegenerative disease, parkinsonism, which is believed to arise from the destruction of dopaminergic neurons, thus greatly lowering brain dopamine levels (40), may involve mitochondrial dysfunction (41). If this indeed results in a rise in the cytosolic levels of Mn 2ϩ in such cells, the activation of M-form PST may help to detoxify the dopamine and possibly other toxic substances (as discussed below) that could be released by such dying cells. In this connection, it may be pertinent to note that manganese poisoning (or manganism) is known to result in symptoms resembling Parkinson's disease (42,43). One reason could be because the activation of M-form PST in dopaminergic cells by the excess Mn 2ϩ results in mis-guided "detoxification" of dopamine in these cells and consequently parkinsonian symptoms.
Physiological Relevance of the Sulfation of D-Tyrosine by M-form PST-Our study has demonstrated M-form PST to be more active toward the D-enantiomers of tyrosine and Dopa. The stimulatory effect of Mn 2ϩ was also much more dramatic FIG. 7. Plots of velocity (v) versus the total concentration of the D-tyrosine-Mn 2؉ plus D-tyrosine-Mn 2؉ -Dtyrosine complexes derived from the data presented in Fig. 2. The concentrations in mol/liter of the complexes are calculated from the concentrations of total Mn 2ϩ and total D-tyrosine in the assay mixture based on the procedure described under "Discussion." v, in units of nmol of product formed/min/mg of protein, is the velocity of the sulfation of D-tyrosine catalyzed by the M-form PST. Each data point represents the mean value of three determinations (error bars are shown). with these D-enantiomers. Although the L-enantiomer of amino acids is used in protein synthesis, a small percentage of the amino acid pool is present in the D-form. D-Amino acids may be formed due to spontaneous racemization in proteins with low turnover rates, such as human lens protein (44), and accumulated in aging tissues lacking D-amino acid oxidases (13). Attempts have been made to link the amount of specific D-amino acid to oxidative damage and to neurodegenerative disorders such as Alzheimer's and Parkinson's disease (14,15). Although a clear picture has yet to emerge, the removal of D-amino acids, which cannot participate in protein synthesis and in most metabolic reactions, may be viewed as a detoxification process. Incidentally, detoxification of D-amino acids through sulfation is likely to be less deleterious than by D-amino acid oxidase, which causes oxidative stress (45). In a different perspective, the Mn 2ϩ -stimulated activity of M-form PST toward D-Dopa and D-tyrosine may provide clues to the understanding of its stereoselective action on chiral drugs (18 -21).
Possibility of a Dual Role of M-form PST with Mn 2ϩ Serving as a Molecular Switch-It is possible that M-form PST under normal circumstances acts on its physiological substrate, dopamine (of which the pH optimum is ϳ7.0, and the K m is ϳ 2 M) (22,46), thereby regulating the levels of this endogenous metabolite. In the presence of elevated Mn 2ϩ (possibly under conditions of oxidative stress, as discussed previously), the detoxifying action of M-form PST is activated. Mn 2ϩ may complex with various substrates, with varying affinities, and these complexes may serve as substrates for M-form PST. Mn 2ϩ may thus serve as a molecular switch to increase the substrate promiscuity of M-form PST. The affinity of the enzyme for these xenobiotic substrates will depend on the dissociation constant of the substrate-metal complex, the metal ion concentration, and the affinity of the enzyme for the complex. Incidentally the different pH optimum (ϳ8 -9 for the Dopa/tyrosine-sulfating activity of M-form PST (8)) compared with the pH optimum of around 7 for dopamine sulfation may reflect the pH dependence of the formation of substrate-metal complex and offer another level of regulation. Because sulfation is an energetically expensive process (it uses up PAPS, synthesis of one molecule of which requires expenditure of three high energy phosphate bonds of ATP (1)), this proposed dual function of M-form PST may make sense from the viewpoint of cellular economy.
In conclusion, our findings on the stimulatory effect of Mn 2ϩ on the sulfation of D-Dopa and D-tyrosine by M-form PST through the formation of a substrate-Mn 2ϩ complex represent the first report of a regulatory mechanism operating in the ST enzymes. It is possible that other xenobiotic substrates may also be acted on by this enzyme in a similar fashion, in concert with Mn 2ϩ or other metal ions, although the affinity and concentrations involved may be quite different depending on the dissociation constant of the metal-substrate complex and other parameters. It would be interesting to see if other examples of such regulatory mechanisms, possibly involving other molecular signals, also operate among other members of this important family of enzymes.