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J. Biol. Chem., Vol. 275, Issue 45, 35070-35076, November 10, 2000

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Mechanism of Phosphoanhydride Cleavage by Baculovirus Phosphatase^*

Alexandra Martins and Stewart Shuman

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Received for publication, June 30, 2000, and in revised form, August 15, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Baculovirus phosphatase (BVP) is a member of the metazoan RNA triphosphatase enzyme family that includes the RNA triphosphatase component of the mRNA capping apparatus. BVP and other metazoan RNA triphosphatases belong to a superfamily of phosphatases that act via the formation and hydrolysis of a covalent cysteinyl-phosphate intermediate. Here we demonstrate the formation of a BVP phosphoenzyme upon reaction with [-³²P]ATP and identify the linkage as a thiophosphate based on its chemical lability. We surmise that the phosphate is linked to Cys¹¹⁹ of BVP because replacement of Cys¹¹⁹ by alanine or serine abrogates phosphoenzyme formation and phosphohydrolase activity. The catalytic cysteine is situated within a conserved phosphate-binding loop (¹¹⁸HCTHGINRTGY¹²⁸). We show that all of the non-aliphatic side chains of the phosphate-binding loop are functionally important, insofar as mutants H118A, H121A, N124A, R125A, T126A, and Y128A were inactive in  phosphate hydrolysis and the T120A mutant was 7% as active as wild-type BVP. Structure-activity relationships at the essential positions of the phosphate-binding loop were elucidated by conservative substitutions. A conserved aspartic acid (Asp⁶⁰) invoked as a candidate general acid catalyst was dispensable for phosphohydrolase activity and phosphoenzyme formation by BVP. We propose that the low pK_a of the bridging oxygen of the  phosphate leaving group circumvents a requirement for expulsion by a proton donor during attack by cysteine on the  phosphorus. In contrast, a conserved aspartic acid is essential for the phosphomonoesterase reactions catalyzed by protein phosphatases, where the serine or tyrosine leaving groups have a much higher pK_a than does ADP.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

RNA triphosphatase catalyzes the hydrolysis of the  phosphate of triphosphate-terminated RNA to form a diphosphate end. Two distinct classes of eukaryotic RNA triphosphatases have been described. The RNA triphosphatases of metazoans and plants belong to a superfamily of phosphatases that includes protein-tyrosine phosphatases, dual specificity protein phosphatases, and phosphoinositide phosphatases (1-12). The metazoan RNA triphosphatases do not require a metal cofactor. In contrast, the RNA triphosphatases of fungi and DNA viruses (poxviruses and baculoviruses), which comprise a new family of nucleoside triphosphate phosphohydrolases, are strictly dependent on a divalent cation cofactor (13-19). There are no mechanistic or structural similarities whatsoever between the metazoan and viral/fungal triphosphatase families (20). Thus, RNA triphosphatase presents a remarkable case of complete divergence during the transition from fungal to metazoan species.

The metazoan RNA triphosphatases are subdivided into two groups: (i) bifunctional enzymes composed of an N-terminal RNA triphosphatase domain and a C-terminal RNA guanylyltransferase domain (1-9) and (ii) monofunctional RNA triphosphatases (10-12). The bifunctional enzymes are ubiquitous in higher eukaryotes, where they are responsible for forming the 5' cap structure of cellular mRNAs. The monofunctional enzymes perform RNA 5' modification reactions uncoupled from or unrelated to cap formation, and their true role in RNA metabolism is not understood. The latter category includes BVP,¹ a phosphatase encoded by the Autographa californica baculovirus (10, 11), and the human PIR1 phosphatase (12). Also in the latter category are the alternatively spliced forms of the cellular capping enzymes that retain the RNA triphosphatase domain but lack essential constituents of the guanylyltransferase active site (7-9).

The RNA triphosphatase domains of the metazoan and plant capping enzymes contain the signature HCXXGXXR(S/T) phosphate-binding motif of the protein-tyrosine phosphatase/dual specificity protein phosphatase enzyme superfamily (Fig. 1). Protein-tyrosine phosphatases and dual specificity protein phosphatases catalyze the dephosphorylation of phosphoproteins via a two-step pathway (21, 22). First, a cysteine thiolate nucleophile of the enzyme attacks the phosphomonoester (R-O-PO₃) to form a covalent protein-cysteinyl-S-phosphate intermediate and liberate R-OH (Fig. 1). Then, the covalent intermediate is hydrolyzed to liberate inorganic phosphate. The cysteine within the signature motif is the active site of phosphoryl transfer by protein phosphatases. The attack of cysteine on a phosphomonoester to form the phosphoenzyme is driven by two principal enzymic catalysts: (i) a conserved arginine in the phosphate-binding motif that makes a bidentate interaction with the nonbridging phosphate oxygens and thereby stabilizes the transition state, and (ii) a conserved aspartate, which is protonated in the ground state and acts as a general acid to expel the leaving group oxygen. The general acid is located within a flexible loop element that is distant from the active site pocket when the enzyme is in the unliganded state and moves into the active site upon binding of substrate or substrate analog (23-26). A mechanism of regulation of protein phosphatases through the flexible general acid loop, either positively by promoting closure of the loop or negatively by hindering its mobility, has been discussed (26).

Although several groups have reported that mutation of the active-site cysteine of metazoan RNA triphosphatases to alanine or serine abolishes RNA triphosphatase activity in vitro and in vivo (1, 4, 6, 10, 11), there is as yet no direct evidence for the formation of a phosphoenzyme during the RNA triphosphatase reaction.

Here we probe the catalytic mechanism of the baculovirus RNA triphosphatase BVP. The 168-amino acid BVP protein displays extensive sequence similarity to the triphosphatase domains of metazoan and plant capping enzymes. The similarity embraces the cysteine-containing signature motif and a single conserved aspartic acid (Fig. 1). Purified recombinant BVP hydrolyzes the  phosphate of triphosphate-terminated poly(A) or the triphosphate-terminated trinucleotide pppApCpC (10, 11). BPV also hydrolyzes ATP to ADP and GTP to GDP (10). The remarkable feature of BVP is that it also catalyzes the hydrolysis of ADP to AMP and GDP to GMP (10) and the conversion of diphosphate-terminated RNA to monophosphate-terminated RNA (11). Thus, BVP is a triphosphatase and a diphosphatase. Kinetic analysis of the reaction of BVP with either triphosphate-terminated RNA or free nucleoside triphosphates shows that the  phosphate is hydrolyzed prior to the  phosphate and that the enzyme acts distributively, i.e. that nucleoside monophosphate products do not begin to accumulate until the majority of the input nucleoside triphosphate has been first converted to nucleoside diphosphate. Human PIR1, like BVP, is also an RNA triphosphatase and diphosphatase (12). In contrast, the metazoan capping enzymes hydrolyze only the  phosphate of RNA. Thus, BVP and PIR1 comprise a functionally distinctive subfamily of cysteinyl phosphatases.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Metazoan RNA triphosphatases. The amino acid sequence of the baculovirus-encoded RNA triphosphatase BVP from amino acids 55 to 134 is aligned with human PIR1 and with the sequences of the N-terminal RNA triphosphatase domains of mouse capping enzyme (Mus CE), C. elegans capping enzyme (Cel CE), Drosophila melanogaster capping enzyme (Dme CE), Xenopus laevis capping enzyme (Xla CE), and two putative capping enzymes of Arabidopsis thaliana (Ath CE1 and CE2). Gaps in the sequences are indicated by dashes (-). The protein phosphatase signature motif is highlighted in the shaded box. The active site cysteine is denoted by an arrow. A conserved Asp residue located proximal to the signal motif is also shaded. The 12 residues of BVP subjected to mutational analysis are denoted by asterisks. The reaction pathway through a cysteinyl phosphate intermediate is shown. For the RNA triphosphatases, R is a diphosphate-terminated RNA.

We demonstrate here the formation of a covalent phosphoenzyme adduct during the reaction of BVP with [-³²P]ATP. Suboptimal reaction conditions, i.e. low temperature and mildly acidic pH, were required to detect the phosphoenzyme. The lability of the phosphoenzyme adduct to treatment with iodine was consistent with a cysteinyl-phosphate adduct. Alanine scanning mutational analysis of the signature phosphatase motif established essential roles for eight side chains in  phosphate cleavage. Structure-activity relationships at the essential positions were then illuminated by conservative substitutions. A remarkable finding was that the conserved upstream aspartate was dispensable for the phosphohydrolase activity of BVP. Mechanistic implications of these results are discussed.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Missense Mutants of BVP-- Plasmid pET16-BVP encodes the 168-amino acid BVP polypeptide fused to an N-terminal leader peptide containing 10 tandem histidines (10). Expression of the protein is under the control of a T7 RNA polymerase promoter. Missense mutations and "silent" diagnostic restriction sites were introduced into the BVP gene by polymerase chain reaction using the two-stage overlap extension method (39). pET16-BVP was used as the template for the first-stage amplifications. The mutated DNA products of the second-stage amplification were digested with NdeI and BamHI and inserted into pET16b. The presence of the desired mutations was confirmed by DNA sequencing; the inserted restriction fragments were sequenced completely in order to exclude acquisition of unwanted mutations during amplification and cloning.

Expression and Purification of Recombinant BVP-- The wild-type and mutated pET16-BVP plasmids were transformed into Escherichia coli BL21. BVP expression was induced by infection of the cells with CE6, which carries the T7 RNA polymerase gene. Cultures (1 liter) of E. coli BL21/pET16-BVP were grown at 37 °C in Luria-Bertani medium containing 0.2% maltose and 0.1 mg/ml ampicillin until the A₆₀₀ reached 0.3-0.4. The cultures were supplemented with 10 mM MgSO₄ and 0.4% glucose, and incubation was continued at 37 °C until the A₆₀₀ reached ~0.8. The cultures were chilled on ice for 15 min and then infected by adding CE6 stock to attain a multiplicity of 5. The cultures were then incubated for 4 h at 25 °C with continuous shaking, after which the cells were harvested by centrifugation. The recombinant BVP proteins were purified from soluble bacterial lysates by nickel-agarose and phosphocellulose column chromatography as described previously (10).

Determination of BVP Protein Concentration-- Aliquots of each phosphocellulose BVP preparation were analyzed by SDS-PAGE in parallel with 1, 2, 3, and 4 µg of bovine serum albumin. The gels were fixed and stained with Coomassie Blue dye. The staining intensity of the polypeptides was quantitated with a Digital Imaging and Analysis System (Alpha Innotech Corp.). BVP concentrations were calculated by extrapolation to the bovine serum albumin standard curve.

Detection and Chemical Stability of a BVP Phosphoenzyme-- A reaction mixture containing 50 mM Tris acetate (pH 5.5), 1 mM DTT, 3 µM BVP, and 20 µM [-³²P]ATP was incubated on ice for 45 s. The mixture was then made 2% in SDS. Aliquots (1.5 µl) were distributed to separate tubes and treated with the following reagents in a total volume of 15 µl: 0.1 M NaOH, 1 M hydroxylamine (pH 7.0), 0.1 M hydroxylamine (pH 7.0), 1 mM iodine (pH 7.0), 1 M Tris-HCl (pH 7.0), 0.1 M Tris-HCl (pH 7.0). Additional aliquots (1.5 µl) were pH-adjusted in a total volume of 15 µl of the following solutions: 1 M HCl, 0.1 M HCl, 0.1 M glycine (pH 2.0), 0.1 M Tris formate (pH 3.0), 0.1 M Tris acetate (pH 4.0, 5.0, or 6.0), Tris-HCl (pH 7.0 or 8.0), 0.1 M BTP (pH 9.0 or 10.0), 0.1 M NaOH. The SDS concentration was maintained at 2% during the chemical treatments. The mixtures were incubated at 37 °C for 30 min, and the samples were analyzed by electrophoresis through a 15% polyacrylamide gel containing 0.1% SDS. (The mixtures in which pH was varied widely from neutrality were adjusted to neutrality prior to SDS-PAGE). The gels were soaked for 30 min in a solution of 3% glycerol and 30% methanol and then dried using the Promega gel-drying kit according to the vendor's instructions. [³²P]BVP adducts were visualized by autoradiography and quantitated by scanning the gel with a Fujix BAS2000 phosphorimager.

ATPase Assay-- Reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.5), 1 mM DTT, 100 µM [-³²P]ATP, and enzyme were incubated for 15 min at 30 °C. The reactions were quenched by adding 2.5 µl of 5 M formic acid. Aliquots of the reaction mixtures were applied to polyethyleneimine cellulose TLC plates that were developed in 0.5 M LiCl, 1 M formic acid. [-³²P]ATP and ³²P_i were quantitated by scanning the TLC plate with a phosphorimager.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Formation of a Covalent Phosphoenzyme Adduct during the Reaction of BVP with ATP-- Although hydrolysis of the  phosphate of triphosphate-terminated RNAs or nucleoside triphosphatases by members of the cysteine phosphatase superfamily is now well documented, there has been no direct evidence presented for a phosphoenzyme intermediate analogous to that shown for the protein-tyrosine phosphatases and dual-specificity protein phosphatases (27, 28). Our initial efforts to trap a ³²P-labeled phosphoenzyme entailed a brief reaction of 3 µM BVP with 3 µM [-³²P]ATP, followed by a quench with SDS. The denatured reaction mixture was analyzed by SDS-PAGE with the expectation of detecting label transfer from ATP to the BVP polypeptide. We were unable to detect a phosphoenzyme when the incubations were performed at 30 °C at pH 7.5 (the optimal conditions for ATP hydrolysis in the steady state) because the substrate was hydrolyzed completely to ³²P_i (data not shown). Decreasing the reaction temperature to 4 °C reduced ATP hydrolysis by BVP by a factor of 18, but did not facilitate detection of a phosphoenzyme at pH 7.5 (data not shown). We inferred that hydrolysis of a putative phosphoenzyme intermediate might be much faster that phosphoryl transfer from ATP when the reactions were performed at pH 7.5. Thus, we varied the pH of the reaction mixture from 3.5 to 9.0. Label transfer from [-³²P]ATP to BVP during a 10-s reaction at 4 °C was detected within a rather narrow window at mildly acidic pH, with optimal trapping of the phosphoenzyme in Tris acetate buffer (pH 5.5) (Fig. 2A). No label transfer was observed when BVP was incubated with [-³²P]ATP (data not shown). Kinetic analysis of the reaction with [-³²P]ATP at pH 5.5 showed that the phosphoenzyme accumulated immediately at 10 s, persisted until 2 min, and decayed from 5 to 30 min (Fig. 2B). The decay of the radiolabeled phosphoprotein was prevented by adding SDS to the reaction mixtures after 30 s of reaction with ATP (data not shown). Thus, the loss of signal did not reflect chemical lability of the phosphoprotein adduct. The finding that phosphoenzyme formation and ATP hydrolysis were both abrogated by mutating the Cys¹¹⁹ to alanine (see below) provided evidence that the phosphoenzyme was relevant to the catalytic mechanism of BVP.

View larger version (64K): [in this window] [in a new window]   Fig. 2.   Formation of a BVP phosphoenzyme. A, pH dependence. Reaction mixtures (10 µl) containing containing 3 µM BVP, 3 µM [-32P]ATP, 1 mM DTT, and 50 mM Tris buffer (Tris formate (pH 3.5), Tris acetate (pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0), or Tris-HCl (pH 7.5, 8.0, 8.5, 9.0)) were incubated on ice for 10 s and then quenched with 2% SDS. The samples were analyzed by SDS-PAGE. An autoradiograph of the wet gel is shown. The positions and sizes (kDa) of pre-stained marker proteins are indicated on the left. B, kinetics of phosphoenzyme formation and decay. A reaction mixture containing 50 mM Tris acetate (pH 5.5), 1 mM DTT, 3 µM BVP, and 3 µM [-32P]ATP was incubated on ice. Aliquots were removed at the times specified and quenched immediately with SDS. The samples were analyzed by SDS-PAGE. An autoradiograph of the wet gel is shown.

Evidence for a Thiophosphate Adduct-- The SDS-denatured [³²P]BVP adduct was stable to 30 min exposure at 37 °C to 0.1 M NaOH, or to 1 M hydroxylamine at pH 7.0, but the ³²P was released quantitatively from the BVP by 1 mM iodine at pH 7.0 (Fig. 3A). Stability to neutral hydroxylamine argues against an acylphosphate linkage, whereas hydrolysis by iodine is strongly indicative of a thiophosphate linkage, presumably to Cys¹¹⁹. A control experiment showed that the phosphoramidate linkage of [³²P]GMP to Lys²⁶⁰ of vaccinia virus capping enzyme was unaffected by treatment with 1 mM iodine (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Chemical stability of the BVP phosphoenzyme. A, [32P]BVP was denatured with SDS and exposed to NaOH, hydroxylamine, or iodine for 30 min at 37 °C. Control samples were exposed to Tris buffer (pH 7.0). The treated samples were analyzed by SDS-PAGE. An autoradiogram of the dried gel is shown. B, the pH stability of [32P]BVP was tested as described under "Experimental Procedures." Samples incubated for 30 min at the indicated pH were analyzed by SDS-PAGE, and the levels of 32P label associated with BVP were quantitated by scanning the dried gel with a phosphorimager. The data were normalized to the amount of residual [32P]BVP at pH 10.0 (defined as 100) and plotted as a function of pH.

The stability of the denatured BVP phosphoenzyme as a function of pH is shown in Fig. 3B. The adduct was alkaline-stable, as noted above, but relatively labile from pH 2 to pH 5. Stability was restored at pH 0 to 1. This trough-like pH stability profile is characteristic of a thiophosphate linkage (29) and has been described previously for the phosphoenzyme of a mammalian protein-tyrosine phosphatase (27).

Mutational Analysis of the Phosphate-binding Loop-- Alanine mutations were introduced in lieu of seven individual amino acids within the conserved phosphate-binding motif of BVP (¹¹⁷VHCTHGINRTGY¹²⁸). The residues mutated were His¹¹⁸, Thr¹²⁰, His¹²¹, Asn¹²⁴, Arg¹²⁵, Thr¹²⁶, and Tyr¹²⁸. Wild-type BVP and the BVP-Ala mutants were expressed in bacteria as His-tagged fusions and purified from soluble bacterial lysates by nickel-agarose and phosphocellulose column chromatography. SDS-PAGE analysis showed that the phosphocellulose preparations were highly enriched with respect to the ~24-kDa BVP polypeptide and that the extents of purification were similar (Fig. 4A). Recombinant wild-type BVP catalyzed the release of ³²P_i from [-³²P]ATP. The extent of ATP hydrolysis was linear with input enzyme at limiting concentrations, and the reaction proceeded to completion at saturating enzyme levels (data not shown). The specific ATPase activities of the BVP-Ala mutants were determined by enzyme titration. The findings are summarized in Fig. 5, where the ATPase activities of the mutants are expressed as the percentage of the wild-type specific activity.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Purification of BVP proteins with mutations of the phosphate-binding loop. Aliquots (1 µg) of the phosphocellulose preparations of wild-type (WT) BVP and the indicated BVP mutants were analyzed by electrophoresis through a 15% polyacrylamide gel containing 0.1% SDS. Polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (in kDa) of marker proteins are indicated. A, alanine mutants; B, conservative mutations.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of phosphate-binding loop mutations on phosphohydrolase activity and phosphoenzyme formation. ATPase activity was assayed as described under "Experimental Procedures." Aliquots (2 µl) of serial 2-fold dilutions of each phosphocellulose BVP preparation were included in the reaction mixtures. Between two and four titration experiments were performed for each protein, and the specific activity was calculated from the average of the slopes of the titration curves. The ATPase specific activities shown are normalized to the specific activity of wild type (WT) BVP (1 pmol of 32Pi released/15 min/ng of protein).

Mutants H118A, H121A, N124A, R125A, T126A, and Y128A were effectively inert in ATP hydrolysis at a level of sensitivity of <0.1% of the wild-type specific activity. These mutational effects were similar to that of the C119A mutation (10). The T120A mutant was 7% as active as wild-type BVP. Thus, all of the non-aliphatic side chains of the phosphate-binding loop were important for activity.

Alanine substitution eliminated the side chain beyond the  carbon, but did not reveal the properties of the missing side chain that were important for activity. This was addressed by introducing conservative substitutions at the seven newly defined essential residues and at Cys¹¹⁹, which was shown previously to be essential for BVP activity (10). The purity of the 15 conservatively substituted recombinant BVP proteins was confirmed by SDS-PAGE (Fig. 4B). The specific ATPase activities of the conservative mutants were determined by enzyme titration and are shown in Fig. 5. The instructive findings are summarized below.

The C119V and C119S proteins were catalytically inert. The failure to restore activity by introducing an isosteric hydroxyamino acid in lieu of cysteine underscored the strict requirement for a thiolate as the reactive nucleophile.

Distinct conservative mutational effects were noted at the two threonine positions of the phosphate-binding loop, Thr¹²⁰ and Thr¹²⁶. The ATPase activity was fully restored when a valine was introduced at Thr¹²⁰; indeed, the T120V mutant was twice as active as wild-type BVP. Valine is nearly isosteric to threonine, but lacks the potential for hydrogen bonding. Placing a serine at position 120 only partially restored activity, i.e. to 26% of the wild-type value, compared with 7% of wild-type activity for the T120A mutant. Given that hydrogen bonding by the hydroxyl was clearly not important, we suspect that the salutary effects of serine at position 120 reflected occupancy by O of one of the two  sites on the threonine/valine side chains. We surmise that two substituents on the  carbon of amino acid 120 are optimal for BVP activity. This contrasts with the situation at position 126, where the T126V mutation was just as deleterious as T126A. However, activity was restored to 65% of the wild-type level by introducing a serine, implying that the hydroxyl group is essential for catalysis, presumably by virtue of its capacity to form a hydrogen bond.

Asn¹²⁴ was replaced conservatively by glutamine and aspartic acid. The N124Q and N124D mutants were as defective in ATP hydrolysis as N124A. These findings suggested that the essential function of Asn¹²⁴ entails hydrogen bonding interactions with the amide moiety that are sensitive to its distance from the main chain.

His¹¹⁸ was changed to valine and asparagine. The H118V and H118N proteins were just as defective as H118A. His¹²¹ was mutated to glutamine and asparagine. H121Q was 2% as active as wild type BVP, whereas H121N was 8% as active. Asparagine and glutamine are partially isosteric with histidine, such that the amide nitrogens of Asn and Gln can be imposed on N and N of His, respectively. The mutational findings at His¹¹⁸ and His¹²¹ of BVP suggested that hydrogen bonding properties of both histidines are important. Interactions of N apparently do not suffice for catalysis at position 118. (We did not construct a H118Q mutant and therefore cannot draw a conclusion concerning N). At position 121, both N and N appeared to be functionally important.

Arg¹²⁵ is conserved in all members of the cysteine phosphatase superfamily. A lysine in lieu of arginine restored phosphohydrolase activity to 10% of the wild-type level. This result suggested that: (i) the positive charge on the side chain is essential for function and (ii) bidentate interactions of the arginine side chain contribute an additional order of magnitude to catalysis.

The essential Tyr¹²⁸ side chain was substituted by phenylalanine and leucine. The Y128F protein was fully active, whereas Y128L was about one-third as active as wild-type BVP. Thus, neither a hydroxyl group nor an aromatic group was strictly essential at this position; rather, a bulky aliphatic side chain sufficed for BVP function.

Mutational Effects on Phosphoenzyme Formation-- The recombinant BVP proteins containing mutations in the phosphate-binding motif were assayed for phosphoenzyme formation during a brief reaction with [-³²P]ATP at pH 5.5. The reaction products were analyzed by SDS-PAGE (Fig. 6). There was good concordance between the mutational effect on steady-state ATP hydrolysis at pH 7.5 and the yield of phosphoenzyme at pH 5.5. For example, the conservative mutants T120V, T120S, R125K, T126S, Y128F, and Y128L that retained ATPase activity were also competent to form the phosphoenzyme. The mutant proteins that were unable to hydrolyze ATP in the steady state were also grossly defective in phosphoenzyme formation. These data provide correlative evidence that the covalent BVP thiophosphate adduct is a genuine reaction intermediate.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Effects of phosphate-binding loop mutations on phosphoenzyme formation. Reaction mixtures containing 50 mM Tris acetate (pH 5.5), 1 mM DTT, 3 µM [-32P]ATP, and 1.7 µM wild-type (WT) or mutant BVP as specified were incubated on ice for 10 s. After quenching with SDS, the mixtures were analyzed by SDS-PAGE. An autoradiogram of the dried gel is shown.

Conserved Asp⁶⁰ Is Dispensable for Catalysis-- Asp⁶⁰, which is conserved in all of the metazoan RNA triphosphatases, is located upstream of the phosphate-binding motif (Fig. 1). A similarly positioned conserved aspartate side chain of the protein-tyrosine phosphatases and dual specificity protein phosphatases functions as a general acid catalyst by donating a proton to the hydroxyamino acid leaving group during the attack of the cysteine nucleophile on the phosphoprotein substrate. Consistent with this role in catalysis, mutations of the aspartate general acid of protein-tyrosine phosphatases, dual specificity protein phosphatases, and phosphoinositide phosphatases to alanine or asparagine result in a drastic loss of catalytic power (22, 30-32). To test the role of Asp⁶⁰ in the phosphohydrolase reaction of BVP, we mutated Asp⁶⁰ to Ala, Asn, and Glu. The purity of the recombinant D60A, D60N, and D60E proteins was confirmed by SDS-PAGE (Fig. 7A). The specific ATPase activities of the D60 mutants were determined by enzyme titration. The remarkable finding was that the D60A and D60N mutations had virtually no effect on ATP hydrolysis (Fig. 7B) or the formation of the [³²P]BVP phosphoenzyme (Fig. 7C). We surmised from this result that hydrolysis of the - phosphoanhydride of ATP by BVP is not limited by the step of expelling the ADP leaving group. Replacing Asp⁶⁰ by glutamate inactivated the enzyme for steady-state ATP hydrolysis and phosphoenzyme formation (Fig. 7). Thus, although the acidic moiety at position 60 is not required for catalysis, there is a strong constraint on the size of the linker arm, such that projection of the carboxylate away from the main chain by an additional methylene group is catastrophic for BVP function.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effects of mutations in the conserved aspartate. A, aliquots (1 µg) of the phosphocellulose preparations of wild-type (WT) BVP and the indicated BVP mutants were electrophoresed through a 15% polyacrylamide gel containing 0.1% SDS. Polypeptides were visualized by staining with Coomassie Blue dye. The positions and sizes (in kDa) of marker proteins are indicated. B, ATPase specific activity and phosphoenzyme formation were assayed as described in the legend to Fig. 5. C, phosphoenzyme formation was assayed as described in the legend to Fig. 6. [32P]BVP was visualized by autoradiography of the dried gel.

An alternative explanation for the nonessentiality of Asp⁶⁰ is that another aspartate residue in BVP serves as the essential proton donor to expel ADP. Phylogenetic considerations made us skeptical of this scenario, insofar as Asp⁶⁰ is the only aspartic acid in BVP that is conserved in the seven other metazoan and plant RNA triphosphatases. Nor are there any glutamate residues of BVP that are conserved in all of the other proteins. Nonetheless, we tested the effects on alanine mutation at Asp⁶⁹ and Asp¹⁰¹, which are the only other aspartates immediately upstream of the BVP phosphate-binding motif (Fig. 1). The D69A mutant was 65% as active as wild-type BVP in ATP hydrolysis, whereas D101A was twice as active as wild-type BVP. Thus, neither Asp⁶⁰ nor Asp¹⁰¹ is essential for catalysis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Here we have initiated an analysis of the catalytic mechanism of BVP, which we regard as a prototype of the metazoan RNA triphosphatase enzyme family that includes the RNA triphosphatase component of the mRNA capping apparatus. We take advantage of the fact that BVP (unlike the metazoan capping enzymes) can use nucleoside triphosphates as substrates for  phosphate cleavage. Thus, we could study BVP and mutated versions thereof using commercially available nucleoside triphosphates instead of triphosphate-terminated RNAs, which are tedious to prepare. The experiments presented here provide insights into BVP catalysis and reveal interesting mechanistic distinctions between the RNA triphosphatases and the protein phosphatase branches of the cysteinyl phosphatase superfamily.

We demonstrate formation of a BVP phosphoenzyme and identify the linkage as a thiophosphate based on its chemical lability. Although we have not directly mapped the location of the phosphoamino acid, it is likely that the phosphate is linked to Cys¹¹⁹ because: (i) replacement of Cys¹¹⁹ by alanine or serine abrogates phosphoenzyme formation, (ii) of the five cysteines in BVP only Cys¹¹⁹ is conserved in all of the metazoan RNA triphosphatases, and (iii) there is definitive evidence that the equivalent cysteine in protein-tyrosine phosphatases and dual specificity protein phosphatases acts as the reactive nucleophile (22, 33).

The cysteine thiolate nucleophile is situated within the defining motif of the cysteine phosphatase superfamily HCXXGXXR(T/S)G. This motif comprises a phosphate-binding loop that facilitates catalysis by protein-tyrosine phosphatases and dual specificity protein phosphatases in two ways. (i) The backbone amides of the loop interact with the cysteine side chain and stabilize it as the reactive thiolate in the ground state, and (ii) the backbone amides and the invariant arginine side chain interact with the phosphate oxygens to activate the bound substrate for nucleophilic attack and to stabilize the trigonal planar transition state. The replacement of the catalytic arginine in Yersinia protein-tyrosine phosphatase by alanine or lysine reduced k_cat by nearly 4 orders of magnitude (34). The failure of the conservative lysine substitution to appreciably restore catalytic power is interpreted as a stringent requirement for the bidentate interaction of two guanidinium nitrogens of this arginine with the equatorial phosphate oxygens in the transition state (22, 33, 34). This model is in keeping with the 5000-fold decrement in k_cat when the Arg residue of human protein-tyrosine phosphatase 1B was replaced by lysine (31). We find that Ala substitution of the BVP arginine reduced phosphohydrolase specific activity by at least 3 orders of magnitude, but, unlike the case of the protein-tyrosine phosphatases, the introduction of a lysine resulted in the recovery of at least 2 orders of magnitude of catalytic power to a level of 10% of wild-type activity. Clearly, the bidentate complex of arginine with the transition state is less critical for BVP catalysis than it is for protein-tyrosine phosphatase. It is possible that another side chain of BVP is in contact with the phosphate and compensates in the transition state for a "second" contact made by the catalytic arginine of protein-tyrosine phosphatase. Alternatively, it may simply be the case that BVP does not fully exploit the bidentate hydrogen-bonding capacity of arginine to accelerate phosphoryl transfer and therefore a monodentate contact of lysine suffices for 10% of the full activity.

We find that the conserved histidine immediately preceding the cysteine nucleophile is essential for catalysis by BVP and that Asn did not function in lieu of His. Substitution of the equivalent histidine of Yersinia protein-tyrosine phosphatase by Asn and Ala reduced k_cat by 2 and 3 orders of magnitude, respectively (35). Replacing the His side chain of human protein-tyrosine phosphatase 1B by alanine reduced k_cat by 2 orders of magnitude (31). The conserved histidine side chain participates in a hydrogen bonding network that serves to stabilize the cysteine thiolate of protein-tyrosine phosphatase (23). Our mutational data are consistent with a similar essential function in BVP.

Thr¹²⁶ is essential for catalysis by BVP and is conserved as a threonine or serine on the protein-tyrosine phosphatases and dual specificity protein phosphatases. The hydroxyl moiety of the side chain is necessary and sufficient for BVP function, insofar as activity is restored when serine in present. Replacing the Thr or Ser residues of protein-tyrosine phosphatases and dual specificity protein phosphatases by alanine lowers k_cat by at least 2 orders of magnitude (36-38). The crystal structures of protein-tyrosine phosphatases and dual specificity protein phosphatases reveal that the O of serine or threonine donates a hydrogen bond to the active site cysteine, an interaction that may stabilize the cysteine thiolate leaving group during hydrolysis of the phosphoenzyme. The hydrogen bond between O and the S of cysteine is visualized in the transition state and the cysteinyl-phosphate intermediate (22, 23, 33). Although it has been reported for protein-tyrosine phosphatases and dual specificity protein phosphatases that elimination of O selectively impedes the hydrolysis reaction of the cysteinyl-phosphate (36, 37), we did not detect formation of the covalent phosphoenzyme by the T126A mutant of BVP. Thus, the hydroxyl may play a relatively greater role the first step of BVP catalysis than it does in the case of protein-tyrosine phosphatases and dual specificity protein phosphatases. BVP appears similar in its utilization of the catalytic threonine to the mammalian low molecular weight protein-tyrosine phosphatase, in which the equivalent serine is proposed to facilitate attack of the cysteine thiolate on the scissile phosphate (38).

A novel finding was that three other side chains within the conserved phosphate-binding motif of BVP (Thr¹²⁰, His¹²¹, and Asn¹²⁴) and one side chain immediately flanking the motif (Tyr¹²⁸) are essential for phosphohydrolase activity. Structural studies have established that the backbone amide nitrogens of the phosphate-binding loop form hydrogen bonds to the phosphate oxygen atoms in the enzyme-substrate complex, the transition state and the cysteinyl-phosphate intermediate (33), but the roles of the side chains of the loop (other than those discussed above) have received little attention because they are not strictly conserved in the protein-tyrosine phosphatases and dual specificity protein phosphatases. However, Thr¹²⁰, His¹²¹, Asn¹²⁴, and Tyr¹²⁸ of BVP are tightly conserved in the other metazoan RNA triphosphatases (Fig. 1). We presume that these essential side chains either: (i) participate directly in substrate binding or catalysis (perhaps His¹²¹ or Asn¹²⁴) or (ii) function indirectly by ensuring the proper conformation of the loop (Thr¹²⁰ or Tyr¹²⁸) in a manner that is specific to the RNA triphosphatase subfamily of cysteinyl phosphatases.

A key finding of the present study was that the conserved aspartate of BVP is dispensable for phosphohydrolase activity and phosphoenzyme formation. This result is in stark contrast to the essential role of aspartate in catalysis by other cysteinyl phosphatases. For example, an alanine substitution of the conserved aspartic acid in human protein-tyrosine phosphatase 1B decreases k_cat by 5 orders of magnitude (31). Replacing the aspartic acid by asparagine, which had no significant effect on BVP, reduced the k_cat of Yersinia protein-tyrosine phosphatase by 3 orders of magnitude (30) and slowed the activity of the human dual specificity protein phosphatase VHR by 2 orders of magnitude (22).

A simple explanation for the benign effects of the D60A and D60N mutations of BVP is that the enzyme is cleaving a phosphoanhydride bond in which the pK_a of ~6.7 for the bridging oxygen of leaving group ADP is sufficiently low that it does not require facilitated expulsion by a proton donor. This is in contrast to the phosphomonoesterase reactions of protein-tyrosine phosphatases and dual specificity protein phosphatases, where the serine leaving group has a pK_a of 14 and the tyrosine leaving group has a pK_a of 10. With protein-tyrosine phosphatases and dual specificity protein phosphatases, the leaving group pK_a has little effect on catalysis because its expulsion is driven by proton donation from the aspartic acid (22, 26). However, when the Asp is replaced by Asn, the k_cat of the mutant enzyme displays a significant dependence on leaving group pK_a, with reactivity being enhanced when the pK_a is low and impeded when pK_a is high (22). Regulated closure of the general acid loop of a wild type dual specificity protein phosphatase enzyme is critical when the pK_a of the leaving group is high, but much less important when the leaving group has a low pK_a (26). To our knowledge, BVP is the first case in which the presumptive "natural" phosphatase substrate (a phosphoanhydride) is cleaved efficiently without the aid of a conserved aspartate general acid.

Finally, it is instructive to compare the mutational effects on BVP to those reported by Wen et al. (6) for the RNA triphosphatase domain of mammalian mRNA capping enzyme. They found, as did we, that replacing the invariant Arg in the phosphate-binding loop by alanine abolished triphosphatase activity. However, they reported that alanine substitutions for conserved residues His¹²⁵ and Thr¹³³ in the phosphate-binding loop of mouse RNA triphosphatase (equivalent to His¹¹⁸ and Thr¹²⁶ of BVP) elicited only 2-fold reductions in triphosphatase activity (6), whereas we find that the same changes abrogated the activity of BVP. As mentioned above, the mutational effects at the His and Thr positions of BVP are quantitatively in good accord with the mutational data for protein-tyrosine phosphatases and dual specificity protein phosphatases. Wen et al. (6) found that an alanine mutation of the conserved aspartic acid of mammalian capping enzyme reduced its triphosphatase activity to 10% of the wild-type level. While this mutation has a greater impact on capping enzyme than BVP, the 10-fold effect on capping enzyme is much less than the 10² to 10⁵ catalytic decrements incurred by mutations of the aspartate general acid in protein-tyrosine phosphatases, dual specificity protein phosphatases, and phosphoinositide phosphatases.

In conclusion, we have highlighted mechanistic similarities between the virus-encoded RNA triphosphatase BVP and other members of the cysteinyl phosphatase superfamily, while illuminating structure-function relationships that are unique to BVP. Further mutational and structural analyses of several different members of the RNA triphosphatase subfamily will be required in order to account for their unique specificity in hydrolyzing phosphoanhydride substrates.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Molecular Biology Program, Sloan-Kettering Inst., 1275 York Ave., New York, NY 10021. E-mail: s-shuman@ski.mskcc.org.

Published, JBC Papers in Press, August 22, 2000, DOI 10.1074/jbc.M005748200

	ABBREVIATIONS

The abbreviations used are: BVP, baculovirus phosphatase; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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