Catalytically promiscuous enzymes are an attractive frontier for biochemistry, because enzyme promiscuities not only plausibly explain enzyme evolution through the mechanism of gene duplication but also could provide an efficient route to changing the catalytic function of proteins by mimicking this evolutionary process. PP1γ is an effectively promiscuous phosphatase for the hydrolysis of both monoanionic and dianionic phosphate ester-based substrates. In addition to its native phosphate monoester substrate, PP1γ catalyzes the hydrolysis of aryl methylphosphonates, fluorophosphate esters, phosphorothioate esters, and phosphodiesters, with second-order rate accelerations that fall within the narrow range of 1011–1013. In contrast to the different transition states in the uncatalyzed hydrolysis reactions of these substrates, PP1γ catalyzes their hydrolysis through similar transition states. PP1γ does not catalyze the hydrolysis of a sulfate ester, which is unexpected. The PP1γ active site is tolerant of variations in the geometry of bound ligands, which permit the effective catalysis even of substrates whose steric requirements may result in perturbations to the positioning of the transferring group, both in the initial enzyme–substrate complex and in the transition state. The conservative mutation of arginine 221 to lysine results in a mutant that is a more effective catalyst toward monoanionic substrates. The surprising conversion of substrate preference lends support to the notion that mutations following gene duplication can result in an altered enzyme with different catalytic capabilities and preferences and may provide a pathway for the evolution of new enzymes.

Catalysis in protein tyrosine phosphatases (PTPs) involves movement of a protein loop called the WPD loop that brings a conserved aspartic acid into the active site to function as a general acid. Mutation of the tryptophan in the WPD loop of the PTP YopH to any other residue with a planar, aromatic side chain (phenylalanine, tyrosine, or histidine) disables general acid catalysis. Crystal structures reveal these conservative mutations leave this critical loop in a catalytically unproductive, quasi-open position. Although the loop positions in crystal structures are similar for all three conservative mutants, the reasons inhibiting normal loop closure differ for each mutant. In the W354F and W354Y mutants, steric clashes result from six-membered rings occupying the position of the five-membered ring of the native indole side chain. The histidine mutant dysfunction results from new hydrogen bonds stabilizing the unproductive position. The results demonstrate how even modest modifications can disrupt catalytically important protein dynamics. Crystallization of all the catalytically compromised mutants in the presence of vanadate gave rise to vanadate dimers at the active site. In W354Y and W354H, a divanadate ester with glycerol is observed. Such species have precedence in solution and are known from the small molecule crystal database. Such species have not been observed in the active site of a phosphatase, as a functional phosphatase would rapidly catalyze their decomposition. The compromised functionality of the mutants allows the trapping of species that undoubtedly form in solution and are capable of binding at the active sites of PTPs, and, presumably, other phosphatases. In addition to monomeric vanadate, such higher-order vanadium-based molecules are likely involved in the interaction of vanadate with PTPs in solution.

LDP3 (VHZ) is the smallest classical protein tyrosine phosphatase (PTP) known to date and was originally misclassified as an atypical dual-specificity phosphatase. Kinetic isotope effects with steady-state and pre-steady-state kinetics of VHZ and mutants with p-nitrophenol phosphate have revealed several unusual properties. VHZ is significantly more active than previously reported but remains one of the least active PTPs. Highly unusual for a PTP, VHZ possesses two acidic residues (E134 and D65) in the active site. D65 occupies the position corresponding to the typical general acid in the PTP family. However, VHZ primarily utilizes E134 as the general acid, with D65 taking over this role when E134 is mutated. This unusual behavior is facilitated by two coexisting, but unequally populated, substrate binding modes. Unlike most classical PTPs, VHZ exhibits phosphotransferase activity. Despite the presence of the Q-loop that normally prevents alcoholysis of the phosphoenzyme intermediate in other classical PTPs, VHZ readily phosphorylates ethylene glycol. Although mutations of Q-loop residues affect this phosphotransferase activity, mutations on the IPD loop that contains the general acid exert more control over this process. A single P68V substitution on this loop completely abolishes phosphotransferase activity. The ability of native VHZ to catalyze transphosphorylation may lead to an imbalance of intracellular phosphorylation, which could explain the correlation of its overexpression with several types of cancer.

Vanadate is a potent modulator of a number of biological processes and has been shown by crystal structures and NMR spectroscopy to interact with numerous enzymes. Although these effects often occur under conditions where oligomeric forms dominate, the crystal structures and NMR data suggest that the inhibitory form is usually monomeric orthovanadate, a particularly good inhibitor of phosphatases because of its ability to form stable trigonal-bipyramidal complexes. We performed a computational analysis of a 1.14 Å structure of the phosphatase VHZ in complex with an unusual metavanadate species and compared it with two classical trigonal-bipyramidal vanadate–phosphatase complexes. The results support extensive delocalized bonding to the apical ligands in the classical structures. In contrast, in the VHZ metavanadate complex, the central, planar VO3– moiety has only one apical ligand, the nucleophilic Cys95, and a gap in electron density between V and S. A computational analysis showed that the V–S interaction is primarily ionic. A mechanism is proposed to explain the formation of metavanadate in the active site from a dimeric vanadate species that previous crystallographic evidence has shown to be able to bind to the active sites of phosphatases related to VHZ. Together, the results show that the interaction of vanadate with biological systems is not solely reliant upon the prior formation of a particular inhibitory form in solution. The catalytic properties of an enzyme may act upon the oligomeric forms primarily present in solution to generate species such as the metavanadate ion observed in the VHZ structure.

The recently discovered 150-residue human VHZ (VH1-related protein, Z member) is one of the smallest protein tyrosine phosphatases (PTPs) known and contains only the minimal structural elements common to all PTPs. We report a substrate screening analysis and a crystal structure of the VHZ complex with vanadate at 1.1 Å resolution, with a detailed structural comparison with other members of the protein tyrosine phosphatase family, including classical tyrosine-specific protein tyrosine phosphatases (PTPs) and dual-specificity phosphatases (DSPs). A screen with 360 phosphorylated peptides shows VHZ efficiently catalyzes the hydrolysis of phosphotyrosine (pY)-containing peptides but exhibits no activity toward phosphoserine (pS) or phosphothreonine (pT) peptides. The new structure reveals a deep and narrow active site more typical of the classical tyrosine-specific PTPs. Despite the high degrees of structural and sequence similarity between VHZ and classical PTPs, its general acid IPD-loop is most likely conformationally rigid, in contrast to the flexible WPD counterpart of classical PTPs. VHZ also lacks substrate recognition domains and other domains typically found on classical PTPs. It is therefore proposed that VHZ is more properly classified as an atypical PTP rather than an atypical DSP, as has been suggested.

The purple acid phosphatases (PAP) are binuclear metallohydrolases that catalyze the hydrolysis of a broad range of phosphomonoester substrates. The mode of substrate binding during catalysis and the identity of the nucleophile is subject to debate. Here, we used native Fe3+−Fe2+ pig PAP (uteroferrin; Uf) and its Fe3+−Mn2+ derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe2+ by Mn2+ lowers the reactivity of Uf. However, using stopped-flow measurements it could be shown that this replacement facilitates approximately a ten-fold faster reaction between both substrate and inorganic phosphate with the chromophoric Fe3+ site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a μ-1,3 phosphate complex. The slower rate of interaction between substrate and the Fe3+ site relative to catalysis suggests that the substrate is hydrolyzed while coordinated only to the divalent metal ion. The likely nucleophile is a water molecule in the second coordination sphere, activated by a hydroxide terminally coordinated to Fe3+. The faster rates of interaction with the Fe3+ site in the Fe3+−Mn2+ derivative than the native Fe3+−Fe2+ form are likely mediated via a hydrogen bond network connecting the first and second coordination spheres, and illustrate how the selection of metal ions may be important in fine-tuning the function of this enzyme.

Reactivities of five phosphonate esters each coordinated to a dinuclear Co(III) complex were investigated ([Co2(tacn)2(OH)2{O2P(Me)OAr}]3+; tacn = 1,4,7-triazacyclononane; substituent = m-F, p-NO2 (1a); p-NO2 (1b); m-NO2 (1c); p-Cl (1d); unsubstituted (1e)). Hydrolysis of the phosphonate esters in 1a to 1e is specific base catalyzed and takes place by intramolecular oxide attack on the bridging phosphonate. These data define a Brønsted βlg of −1.12, considerably more negative than that of the hydrolysis of the uncomplexed phosphonates (−0.69). For 1b, the kinetic isotope effects in the leaving group are 18klg = 1.0228 and 15k = 1.0014, at the nonbridging phosphoryl oxygens 18knonbridge = 0.9954, and at the nucleophilic oxygen18knuc = 1.0105. The KIEs and the βlg data point to a transition state for the alkaline hydrolysis of 1b that is similar to that of a phosphate monoester complex with the same leaving group, rather than the isoelectronic diester complex. The data from these model systems parallel the observation that in protein phosphatase-1, which has an active site that resembles the structures of these complexes, the catalyzed hydrolysis of aryl methylphosphonates and aryl phosphates are much more similar to one another than the uncomplexed hydrolysis reactions of the two substrates.

The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKa values of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Brønsted βlg of −0.32, considerably less negative than that of the uncatalyzed hydrolysis of monoester dianions (−1.23). Kinetic isotope effects in the leaving group with the substrate 4NPP are 18(V/K)bridge = 1.0170 and 15(V/K) = 1.0010, which, compared against other enzymatic KIEs with and without general acid catalysis, are consistent with a loose transition state with partial neutralization of the leaving group. PP1 also efficiently catalyzes the hydrolysis of 4-nitrophenyl methylphosphonate (4NPMP). The enzymatic hydrolysis of a series of aryl methylphosphonate substrates yields a Brønsted βlg of −0.30, smaller than the alkaline hydrolysis (−0.69) and similar to the βlg measured for monoester substrates, indicative of similar transition states. The KIEs and the βlg data point to a transition state for the alkaline hydrolysis of 4NPMP that is similar to that of diesters with the same leaving group. For the enzymatic reaction of 4NPMP, the KIEs are indicative of a transition state that is somewhat looser than the alkaline hydrolysis reaction and similar to the PP1-catalyzed monoester reaction. The data cumulatively point to enzymatic transition states for aryl phosphate monoester and aryl methylphosphonate hydrolysis reactions that are much more similar to one another than the nonenzymatic hydrolysis reactions of the two substrates.