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Protein tyrosine phosphatases (PTPs) possess a conserved mobile catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. We have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics. The chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, and show differences in the pH dependency of catalysis, and changes in the effect of Mg2+. The chimeric proteins’ WPD-loops differ significantly in their relative stability and rigidity. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.

Conformational dynamics are important factors in the function of enzymes, including protein tyrosine phosphatases (PTPs). Crystal structures of PTPs first revealed the motion of a protein loop bearing a conserved catalytic aspartic acid, and subsequent nuclear magnetic resonance and computational analyses have shown the presence of motions, involved in catalysis and allostery, within and beyond the active site. The tyrosine phosphatase from the thermophilic and acidophilic Sulfolobus solfataricus (SsoPTP) displays motions of its acid loop together with dynamics of its phosphoryl-binding P-loop and the Q-loop, the first instance of such motions in a PTP. All three loops share the same exchange rate, implying their motions are coupled. Further evidence of conformational flexibility comes from mutagenesis, kinetics, and isotope effect data showing that E40 can function as an alternate general acid to protonate the leaving group when the conserved acid, D69, is mutated to asparagine. SsoPTP is not the first PTP to exhibit an alternate general acid (after VHZ and TkPTP), but E40 does not correspond to the sequence or structural location of the alternate general acids in those precedents. A high-resolution X-ray structure with the transition state analogue vanadate clarifies the role of the active site arginine R102, which varied in structures of substrates bound to a catalytically inactive mutant. The coordinated motions of all three functional loops in SsoPTP, together with the function of an alternate general acid, suggest that catalytically competent conformations are present in solution that have not yet been observed in crystal structures.

The two constitutional isomers of naphthyl phosphate have different steric properties, analogous to those of phosphotyrosine versus phosphoserine/threonine within a peptide or protein. The ratios of their respective rates of hydrolysis, assayed by measuring rates of inorganic phosphate release, have been used to probe the steric requirements around the active sites of many phosphatases in the literature. We report an NMR-based competitive method that is simpler to execute and has other advantages. It directly yields the ratio of catalytic efficiencies (V/K) of the two substrates, a more biologically relevant comparison than the ratio of initial rates (v

Protein tyrosine phosphatases (PTPs) possess a mobile, conserved catalytic loop, the WPD-loop, which brings an aspartic acid into the active site where it acts as an acid/base catalyst. Prior experimental and computational studies, focused on the human enzyme PTP1B and the PTP from Yersinia pestis, YopH, suggested that loop conformational dynamics are important in regulating both catalysis and evolvability. Also, work on Chimeras of YopH bearing parts of the WPD-loop sequence from PTP1B demonstrated unusual structural perturbations and reduced activity. In the present study, we have generated a chimeric protein in which the WPD-loop of YopH is transposed into PTP1B, and eight chimeras that systematically restored the loop sequence back to native PTP1B. Of these, four chimeras were soluble and were subjected to detailed biochemical and structural characterization, and a computational analysis of their WPD-loop dynamics in catalysis. These chimeras maintain backbone structural integrity, with somewhat slower rates than either wild-type parent, despite unaltered chemical mechanisms and transition states. The chimeric proteins’ WPD-loops differ significantly in their relative stability and rigidity. In particular, the open WPD-loops sample multiple metastable and interconverting conformations. The time required for interconversion, coupled with electrostatic effects revealed by simulations, likely accounts for the activity differences between chimeras, and relative to the native enzymes. These differences in loop dynamics affect both the pH dependency of catalysis and turnover rate. Our results further the understanding of connections between enzyme activity and the dynamics of catalytically important groups, particularly the effects of non-catalytic residues on key conformational equilibria.

Pseudomonas aeruginosa arylsulfatase (PAS) hydrolyzes sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and analyses of active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs PASWT has a much less negative Brønsted coefficient (βleaving groupobs-Enz = -0.33) than the uncatalyzed reaction (βleaving groupobs = -1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the transition state (TS) is evidenced by an 18Obridge KIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active site K375, in cooperation with H211. 15N KIEs and the increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving groupH-D = +0.06) suggest that the mechanism for S-Obridge bond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions toward direct proton donation. 18Ononbridge KIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate esters by this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

Catalysis by protein tyrosine phosphatases (PTPs) relies on the motion of a flexible protein loop (the WPD-loop) that carries a residue acting as a general acid/base catalyst during the PTP-catalyzed reaction. The orthogonal substitutions of a non-catalytic residue in the WPD-loops of YopH and PTP1B results in shifted pH-rate profiles, from an altered kinetic pKa of the nucleophilic cysteine. Compared to WT, the G352T YopH variant has a broadened pH-rate profile, similar activity at optimal pH, but significantly higher activity at low pH. Changes in the corresponding PTP1B T177G variant are more modest and in the opposite direction, with a narrowed pH profile and less activity in the most acidic range. Crystal structures of the variants show no structural perturbations, but suggest an increased preference for the WPD-loop closed conformation. Computational analysis confirms a shift in loop conformational equilibrium in favor of the closed conformation, arising from a combination of increased stability of the closed state and destabilization of the loop-open state. Simulations identify the origins of this population shift, revealing differences in the flexibility of the WPD-loop and neighboring regions. Our results demonstrate that changes to the pH dependency of catalysis by PTPs can result from small changes in amino acid composition in their WPD-loops affecting only loop dynamics and conformational equilibrium. The perturbation of kinetic pKa values of catalytic residues by non-chemical processes affords a means for nature to alter an enzyme’s pH dependency by a less disruptive path than altering electrostatic networks around catalytic residues themselves.

Protein tyrosine phosphatases (PTPs) play an important role in cellular signaling and have been implicated in human cancers, diabetes, and obesity. Despite shared catalytic mechanisms and transition states for the chemical steps of catalysis, catalytic rates within the PTP family vary over several orders of magnitude. These rate differences have been implied to arise from differing conformational dynamics of the closure of a protein loop, the WPD-Ioop, which carries a catalytically critical residue. The present work reports computational studies of the human protein tyrosine phosphatase 1B (PTP1B) and YopH from Yersinia pestis, for which NMR has demonstrated a link between their respective rates of WPD-Ioop motion and catalysis rates, which differ by an order of magnitude. We have performed detailed structural analysis, both conventional and enhanced sampling simulations of their loop dynamics, as well as empirical valence bond simulations of the chemical step of catalysis. These analyses revealed the key residues and structural features responsible for these differences, as well as the residues and pathways that facilitate allosteric communication in these enzymes. Curiously, our wild-type YopH simulations also identify a catalytically incompetent hyper-open conformation of its WPD-loop, sampled as a rare event, previously only experimentally observed in YopH-based chimeras. The effect of differences within the WPD-loop and its neighboring loops on the modulation of loop dynamics, as revealed in this work, may provide a facile means for the family of PTP enzymes to respond to environmental changes and regulate their catalytic activities. Correction in: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Volume 144, Issue 22, Page 10091-10093, DOI 10.1021/jacs.2c04624

Organophosphorus flame-retardants are stable toxic compounds used in nearly all durable plastic products and are considered major emerging pollutants. The phosphotriesterase from Sphingobium sp. TCM1 (Sb-PTE) is one of the few enzymes known to be able to hydrolyze organophosphorus flame-retardants such as triphenyl phosphate and tris(2-chloroethyl) phosphate. The effectiveness of Sb-PTE for the hydrolysis of these organophosphates appears to arise from its ability to hydrolyze unactivated alkyl and phenolic esters from the central phosphorus core. How Sb-PTE is able to catalyze the hydrolysis of the unactivated substituents is not known. To interrogate the catalytic hydrolysis mechanism of Sb-PTE, the pH-dependence of the reaction and the effects of changing the solvent viscosity were determined. These experiments were complemented by measurement of the primary and secondary 18-oxygen isotope effects on substrate hydrolysis and a determination of the effects of changing the pK(a) of the leaving group on the magnitude of the rate constants for hydrolysis. Collectively, the results indicated that a single group must be ionized for nucleophilic attack and that a separate general acid is not involved in protonation of the leaving group. The Brønsted analysis and the heavy atom kinetic isotope effects are consistent with an early associative transition state with subsequent proton transfers not being rate limiting. A novel binding mode of the substrate to the binuclear metal center and a catalytic mechanism are proposed to explain the unusual ability of Sb-PTE to hydrolyze unactivated esters from a wide range of organophosphate substrates.

After 3 years of laboratory drug pressure in the presence of a picomolar inhibitor, the parasite Plasmodium falciparum developed a combination strategy of gene amplification and mutation to regain viability. The mutation observed led to a dysfunctional enzyme, but new research reveals the clever mechanism behind its success. Not that we needed a reminder of nature’s creativity in the time of a pandemic.

Phosphoimidazole-containing compounds are versatile players in biological and chemical processes. We explore catalytic and mechanistic criteria for the efficient formation of cyclic aryl phosphoimidazoles in aqueous solution, viewed as a template reaction for the in situ synthesis of related compounds. To provide a detailed analysis for this reaction a series of o-(2′-imidazolyl)naphthyl (4-nitrophenyl) phosphate isomers were examined to provide a basis for analysis of both mechanism and the influence of structural factors affecting the nucleophilic attack of the imidazolyl group on the phosphorus center of the substrate. Formation of the cyclic aryl phosphoimidazoles was probed by NMR and ESI-MS techniques. Kinetic experiments show that cyclization is faster under alkaline conditions, with an effective molarity up to 2900 M for the imidazolyl group, ruling out competition from external nucleophiles. Heavy atom isotope effect and computational studies show that the reaction occurs through a SN2(P)-type me...

Salicylate hydroxylase (NahG) is a flavin-dependent monooxygenase that catalyzes the decarboxylative hydroxylation of salicylate into catechol in the naphthalene degradation pathway in Pseudomonas putida G7. We explored the mechanism of action of this enzyme in detail using a combination of structural and biophysical methods. NahG shares many structural and mechanistic features with other versatile flavin-dependent monooxygenases, with potential biocatalytic applications. The crystal structure at 2.0 A resolution for the apo form of NahG adds a new snapshot preceding the FAD binding in flavin-dependent monooxygenases. The kcat/Km for the salicylate reaction catalyzed by the holo form is >105 M−1 s−1 at pH 8.5 and 25 °C. Hammett plots for Km and kcat using substituted salicylates indicate change in rate-limiting step. Electron-donating groups favor the hydroxylation of salicylate by a peroxyflavin to yield a Wheland-like intermediate, whereas the decarboxylation of this intermediate is faster for electron-withdrawing groups. The mechanism is supported by structural data and kinetic studies at different pHs. The salicylate carboxyl group lies near a hydrophobic region that aids decarboxylation. A conserved histidine residue is proposed to assist the reaction by general base/general acid catalysis.

To study factors that affect WPD-loop motion in protein tyrosine phosphatases (PTPs), a chimera of PTP1B and YopH was created by transposing the WPD loop from PTP1B to YopH. Several subsequent mutations proved to be necessary to obtain a soluble, active enzyme. That chimera, termed chimera 3, retains productive WPD-loop motions and general acid catalysis with a pH dependency similar to that of the native enzymes. Kinetic isotope effects show the mechanism and transition state for phosphoryl transfer are unaltered. Catalysis of the chimera is slower than that of either of its parent enzymes, although its rate is comparable to those of most native PTPs. X-ray crystallography and nuclear magnetic resonance were used to probe the structure and dynamics of chimera 3. The chimera's structure was found to sample an unproductive hyper-open conformation of its WPD loop, a geometry that has not been observed in either of the parents or in other native PTPs. The reduced catalytic rate is attributed to the protein's sampling of this conformation in solution, reducing the fraction in the catalytically productive loop-closed conformation.

Pseudomonas aeruginosaarylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present an investigation based on linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs wild type PAS has a much less negative Br0nsted coefficient (βleaving groupobs-Enz= −0.33) than the uncatalyzed reaction (βleavingroupobs= −1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the TS is evidenced by an18ObridgeKIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active-site lysine K375, in cooperation with histidine H211.15N KIEs combined with an increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving groupH-D= +0.06) suggest that the mechanism for S-Obridgebond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions towards direct proton donation.18OnonbridgeKIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate in this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

Phosphoryl transfer is the name given to the chemical process of the transfer of the phosphoryl group (PO3) from a phosphate ester or anhydride to a nucleophile. Nucleophilic attack by water on a phosphate monoester gives the hydrolysis product inorganic phosphate. This net dephosphorylation reaction is the process catalysed by phosphatases. Although the reaction is thermodynamically favourable, it has a very high kinetic barrier, making the uncatalysed hydrolysis of phosphate esters extremely slow. The opposite process, the formation of phosphate esters, is termed phosphorylation and is accomplished in biological systems by kinases. The balance between phosphatase and kinase activities in biology serves to regulate the phosphorylation level of many enzymes and other proteins in the cell, forming an important regulatory mechanism that is found throughout the biological world. Key Concepts The post-translational phosphorylation levels of proteins are regulated by the complimentary actions of phosphatases and kinases. The uncatalysed hydrolysis of phosphate esters has a very high kinetic barrier, making phosphatases among the most efficient enzymes known. The various families of phosphatases use different catalytic machinery and mechanisms to carry out phosphate ester hydrolysis. Keywords: phosphate ester; phosphatase; kinase; phosphorylation; signal transduction

The protein tyrosine phosphatases (PTPs) are a large family of enzymes responsible for intracellular dephosphorylation. Together with protein tyrosine kinases (PTKs), PTPs control the level of protein phosphorylation, which modulates numerous aspects of cell life, such as growth, proliferation, metabolism, intercellular interaction, immune responses, and gene transcription (1). PTPs contain a highly conserved HCXXGXXRS/T signature sequence motif but share very little sequence similarities outside of the conserved regions, which are comprised of the phosphate binding loop (P-loop); the general acid loop, often referred to as the WPD-loop; and the Q-loop that bears conserved glutamine residues that orient the water nucleophile in classical PTPs and prevent phosphotransferase activity to other potential nucleophiles. All PTPs utilize a two-step double-displacement mechanism of phosphate monoester hydrolysis (Scheme 1) mediated by an invariant cysteine-arginine-aspartic acid triad of catalytic residues (2). The mechanism proceeds through a phosphoenzyme intermediate where the second chemical step is often rate limiting (3). In the first step the P-loop orients the substrate as the nucleophilic cysteine attacks phosphorus with simultaneous expulsion of the leaving group protonated by the catalytic general acid. In the second step a water molecule, directed by the aspartic acid residue that served as the general acid in the first step and Q-loop glutamine residues, attacks the phosphoenzyme intermediate. Scheme 1 Top, the chemical steps in the reaction catalyzed by PTPs. In the first chemical step a nucleophilic cysteine attacks the phosphate ester with simultaneous protonation of the leaving group by the conserved aspartic acid. In the second chemical step water ... The PTP family is subdivided into several groups based on substrate specificity, subcellular localization, and size. The classical PTP family selectively hydrolyzes phosphotyrosine containing peptides, and includes the well-studied bacterial effector protein YopH, responsible for the virulence of notorious Y.pestis, and human PTP1B, which plays an important role in insulin signaling (4). Classical PTPs have a modular organization and, in addition to the catalytic phosphatase domain, contain non-catalytic domains that control subcellular localization and protein-substrate interactions. All classical PTPs are tyrosine specific enzymes. The members of the dual-specificity phosphatases (DSPs) subfamily hydrolyze phosphoserine and phosphothreonine in addition to phosphotyrosine containing target sites. Within the DSP subfamily, the atypical DSPs are smaller and contain only a catalytic domain (5). The classical PTPs and DSPs also differ in their phosphotransferase ability. In classical PTPs the phosphoenzyme intermediate is attacked only by water due to the shielding effect of conserved Q-loop residues, named for the presence of conserved glutamines (6). In contrast, DSPs such as VHR, and the low-molecular weight LMW-Ltp1, both of which lack the Q-loop, display significant phosphotransferase ability (7). On this basis, it has been concluded that the presence of the Q-loop prevents phosphotransferase activity. VHZ, and the closely related phosphatase S.solfataricus PTP (SsoPTP), are among the smallest classical PTPs known to date (Figure 1). The SsoPTP (161 amino acids) is similar to VHZ (150 amino acids) in size and catalytic activity. Both VHZ and SsoPTP consist of a single, catalytic domain that is more similar to classical PTPs than DSPs (8, 9) and contain identical secondary structural elements, but, unlike most classical PTPs, lack an N-terminal extension that forms a substrate recognition/binding loop. Like VHZ, the general acid in SsoPTP resides on a rigid IPD-loop, which, unlike the flexible WPD-loop in classical PTPs, permanently occupies a closed conformation. Unlike VHZ, and like classical PTPs, SsoPTP/WT contains no additional general acid in its Q-loop region. VHZ was originally classified as an atypical DSP and named after its prototypical member as VH1-related protein member Z. In previous work, we presented results indicating that VHZ should be classified as a PTP rather than a DSP, on the basis of a structural analysis and results of a phosphopeptide substrate screen in which VHZ showed activity against pY–containing peptides but not toward pS- or pT-peptides (8). Figure 1 Side by side comparison of (A) VHZ/PTP (PDB ID 4ERC), and (B), SsoPTP (PDB ID 2I6J). The proteins are very similar in size and structure, and both contain a rigid IPD-loop (highlighted in red) in contrast to the conserved WPD-loop in classical PTPs. Both ... In the present work, we show that the catalytic activity of VHZ was significantly underestimated in previous reports, as a result of pronounced product inhibition, and the inhibitory effect of certain buffers. Despite much in common with classical PTPs, VHZ is highly unusual in possessing two acidic residues in the active site, D65 and E134. Our results indicate that under certain circumstances either of these residues can serve as the general acid in the first step of the reaction. We also present results demonstrating that VHZ, despite the presence of a Q-loop, catalyzes phosphoryl transfer to alcohols (alcoholysis) in addition to water (hydrolysis) (Scheme 2). Scheme 2 Partitioning of the enzyme-phosphate intermediate [E-P] between hydrolysis and alcoholysis pathways. Alcohols, or a water nucleophile, in two competing pathways attack the phosphoenzyme intermediate formed in the first step. The mutagenesis of several residues in VHZ in parallel with SsoPTP has revealed that, in addition to the Q-loop, particular residues in the general acid IPD-loop play a crucial role in nucleophilic selectivity. A combination of kinetics and mutagenesis experiments have revealed unusual aspects of the kinetic behavior of VHZ and given insights into factors that control the phosphotransferase activity of VHZ, and possibly in other PTPs.

Closing the Loop Many studies have shown that protein dynamics are important to enzyme function. For example, enzyme protein movements have been shown to optimize the active site, enable binding of substrate and cofactor, and facilitate product release. Whittier et al. (p. 899 ) now show that in two tyrosine phosphatases, the rate of cleavage is coupled to motion of a loop. The two phosphatases have different catalytic rates; however, in both, a loop containing a catalytic residue switches between an inactive open and a catalytically competent closed state. The rates of closure are equivalent to the cleavage rates, suggesting that the leaving group tyrosine is protonated simultaneously with loop closure. Thus, tuning of the loop motion plays a regulatory role in the catalytic cycle.

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

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 movement of a conserved protein loop (the WPD-loop) is important in catalysis by protein tyrosine phosphatases (PTPs). Using kinetics, isotope effects, and X-ray crystallography, the different effects arising from mutation of the conserved tryptophan in the WPD-loop were compared in two PTPs, the human PTP1B, and the bacterial YopH from Yersinia. Mutation of the conserved tryptophan in the WPD-loop to phenylalanine has a negligible effect on k(cat) in PTP1B and full loop movement is maintained. In contrast, the corresponding mutation in YopH reduces k(cat) by two orders of magnitude and the WPD loop locks in an intermediate position, disabling general acid catalysis. During loop movement the indole moiety of the WPD-loop tryptophan moves in opposite directions in the two enzymes. Comparisons of mammalian and bacterial PTPs reveal differences in the residues forming the hydrophobic pocket surrounding the conserved tryptophan. Thus, although WPD-loop movement is a conserved feature in PTPs, differences exist in the molecular details, and in the tolerance to mutation, in PTP1B compared to YopH. Despite high structural similarity of the active sites in both WPD-loop open and closed conformations, differences are identified in the molecular details associated with loop movement in PTPs from different organisms.

Catalysis by protein-tyrosine phosphatase 1B (PTP1B) occurs through a two-step mechanism involving a phosphocysteine intermediate. We have solved crystal structures for the transition state analogs for both steps. Together with previously reported crystal structures of apo-PTP1B, the Michaelis complex of an inactive mutant, the phosphoenzyme intermediate, and the product complex, a full picture of all catalytic steps can now be depicted. The transition state analog for the first catalytic step comprises a ternary complex between the catalytic cysteine of PTP1B, vanadate, and the peptide DADEYL, a fragment of a physiological substrate. The equatorial vanadate oxygen atoms bind to the P-loop, and the apical positions are occupied by the peptide tyrosine oxygen and by the PTP1B cysteine sulfur atom. The vanadate assumes a trigonal bipyramidal geometry in both transition state analog structures, with very similar apical O–O distances, denoting similar transition states for both phosphoryl transfer steps. Detailed interactions between the flanking peptide and the enzyme are discussed.

The newly discovered SpvC effector protein from Salmonella typhimurium interferes with the host immune response by dephosphorylating mitogen-activated protein kinases (MAPKs) with a beta-elimination mechanism. To understand this unique phosphothreonine lyase catalysis, the dynamics of the enzyme-substrate complex of the SpvC effector is investigated with a 3.2 ns molecular dynamics simulation, which reveals that the phosphorylated peptide substrate is tightly held in the active site by a hydrogen bond network and the lysine general base is positioned for the abstraction of the alpha hydrogen. The catalysis is further modeled with density functional theory (DFT) in a truncated active-site model at the B3LYP/6-31+G(d,p) level of theory. The DFT calculations indicate the reaction proceeds via a single transition state, featuring a concerted proton abstraction from the alpha-carbon by Lys136 and beta-elimination of the phosphate leaving group. Key kinetic isotopic effects are predicted based on the truncated active-site model.

The cyclization of 2-(hydroxypropyl)-4-nitrophenyl phosphate (HpPNP) catalyzed by the dinuclear zinc complex of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane (1) proceeds by a transition state that is different from that of the uncatalyzed reaction. Kinetic isotope effects (KIEs) measured in the nucleophilic atom and in the leaving group show that the uncatalyzed cyclization has a transition state (TS) with little phosphorus-oxygen bond fission to the leaving group ((18)k(lg) = 1.0064 +/- 0.0009 and (15)k = 1.0002 +/- 0.0002) and that nucleophilic bond formation occurs in the rate-determining step ((18)k(nuc) = 1.0326 +/- 0.0008). In the catalyzed reaction, larger leaving group isotope effects ((18)k(lg) = 1.0113 +/- 0.0005 and (15)k = 1.0015 +/- 0.0005) and a smaller nucleophile isotope effect ((18)k(nuc) = 1.0116 +/- 0.0010) indicate a later TS with greater leaving group bond fission and greater nucleophilic bond formation. These observed nucleophile KIEs are the combined effect of the equilibrium effect on deprotonation of the 2'-hydroxyl nucleophile and the KIE on the nucleophilic step. An EIE of 1.0245 for deprotonation of the hydroxyl group of HPpNP was obtained computationally. The different KIEs for the two reactions indicate that the effective catalysis by 1 is accompanied by selection for an altered transition state, presumably arising from the preferential stabilization by the catalyst of charge away from the nucleophile and toward the leaving group. These results demonstrate the potential for a catalyst using biologically relevant metal ions to select for an altered transition state for phosphoryl transfer.

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 beta(lg) 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 beta(lg) of -0.30, smaller than the alkaline hydrolysis (-0.69) and similar to the beta(lg) measured for monoester substrates, indicative of similar transition states. The KIEs and the beta(lg) 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.

The effects of sulfur substitution on the reactions of hydroxyalkyl phosphate esters are examined. These compounds are models for the intramolecular phosphoryl transfer reaction involved in the cleavage of the internucleotide bond in RNA. The models studied here lack the ribose ring and their conformational flexibility results in greater stability and the availability of different reaction pathways. Sulfur in the nucleophilic position shows no nucleophilic reaction at phosphorus, instead rapidly attacking at the beta carbon atom, forming thiirane with departure of a phosphomonoester. Sulfur substitution at either of the two bridging positions leads to cleavage of the diester via formation of a cyclic intermediate, but with significant rate acceleration when compared to the oxygen analogues. The bridge-substituted models react substantially slower than the analogous ribose compounds with sulfur substitution at comparable positions. Kinetic isotope effects reveal significant differences in the transition state depending on which bridging position sulfur occupies. When sulfur is in the scissile bridging position, a highly associative transition state is indicated, with a largely formed bond to the nucleophile and the scissile P-S bond is little changed. When sulfur occupies the other bridging position, the isotope effects imply a very early transition state in a concerted reaction.

Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis.

Acyl, phosphoryl, and sulfuryl transfer reactions involving esters and amides are ubiquitous in the biochemical world, and these reactions are found throughout the biochemical pathways involving proteins, nucleic acids, and other compounds essential for life. Such compounds are also common in commercial chemical processes. The use of isotopes has contributed greatly to our understanding of the chemistry of these compounds. There is an enormous body of literature on the use of isotopes in such studies, and this review does not do justice to this great body of knowledge. We cover a selection of reactions for which multiple experimental applications using isotopes have been used in the advancement of mechanistic studies. Copyright © 2007 John Wiley & Sons, Ltd.

© 2015 American Chemical Society. 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.

Information about the transition states of metal-catalyzed hydrolysis reactions of model phosphate compounds has been obtained through determination of isotope effects (IEs) on the hydrolysis reactions. Metal complexation has been found to significantly alter the transition state of the reaction from the alkaline hydrolysis reaction, and the transition state is quite dependent on the particular metal ion used. For the diester, ethyl p-nitrophenyl phosphate, the nonbridge 18O effect for the hydrolysis reactions catalyzed by Co(III) 1,5,9-triazacyclononane and Eu(III) were 1.0006 and 1.0016, respectively, indicative of a slightly associative transition state and little net change in bonding to the nonbridge oxygen. The reaction catalyzed by Zn(II) 1,4,7,10-tetraazacyclododecane had an 18O nonbridge IE of 1.0108, showing the reaction differs significantly from the reaction of the noncomplexed diester and resembles the reactions of triesters. Reaction with Co(III) 1,4,7,10-tetraazacyclododecane showed an inverse effect of 0.9948 reflecting the effects of bonding of the diester to the Co(III). Lanthanide-catalyzed hydrolysis has been observed to have unusually large 15N effects. To further investigate this effect, the 15N effect on the reaction catalyzed by Ce(IV) bis-Tris propane solutions at pH 8 was determined to be 1.0012. The 15N effects were also measured for the reaction of the monoester p-nitrophenyl phosphate by Ce(IV) bis-Tris propane (1.0014) and Eu(III) bis-Tris propane (1.0012). These smaller effects at pH 8 indicate that a smaller negative charge develops on the nitrogen during the hydrolysis reaction.

Kinetic isotope effects have been measured for the estrogen sulfotransferase-catalyzed sulfuryl (SO3) transfer from p-nitrophenyl sulfate to the 5′-phosphoryl group of 3′-phosphoadenosine 5′-phosphate. 18(V/K)nonbridge = 1.0016 ± 0.0005, 18(V/K)bridge = 1.0280 ± 0.0006, and 15(V/K) = 1.0014 ± 0.0004. (15(V/K) refers to the nitro group in p-nitrophenyl sulfate). The kinetic isotope effects indicate substantial SO bond fission in the transition state, with partial charge neutralization of the leaving group. The small kinetic isotope effect in the nonbridging sulfuryl oxygen atoms suggests no significant change in bond orders of these atoms occurs, consistent with modest nucleophilic involvement. A comparison of the data for enzymatic and uncatalyzed sulfuryl transfer reactions suggests that both proceed through very similar transition states.

Dual-specificity phosphatases (DSPs) belong to the large family of protein tyrosine phosphatases that contain the active-site motif (H/V)CxxGxxR(S/T), but unlike the tyrosine-specific enzymes, DSPs are able to catalyze the efficient hydrolysis of both phosphotyrosine and phosphoserine/threonine found on signaling proteins, as well as a variety of small-molecule aryl and alkyl phosphates. It is unclear how DSPs accomplish similar reaction rates for phosphoesters, whose reactivity (i.e., pK(a) of the leaving group) can vary by more than 10(8). Here, we utilize the alkyl phosphate m-nitrobenzyl phosphate (mNBP), leaving-group pK(a) = 14.9, as a physiological substrate mimic to probe the mechanism and transition state of the DSP, Vaccinia H1-related (VHR). Detailed pH and kinetic isotope effects of the V/K value for mNBP indicates that VHR reacts with the phosphate dianion of mNBP and that the nonbridge phosphate oxygen atoms are unprotonated in the transition state. (18)O and solvent isotope effects indicate differences in the respective timing of the proton transfer to the leaving group and P-O fission; with the alkyl ester substrate, protonation is ahead of P-O fission, while with the aryl substrate, the two processes are more synchronous. Kinetic analysis of the general-acid mutant D92N with mNBP was consistent with the requirement of Asp-92 in protonating the ester oxygen, either in a step prior to significant P-O bond cleavage or in a concerted but asynchronous mechanism in which protonation is ahead of P-O bond fission. Collectively, the data indicate that VHR and likely all DSPs can match leaving-group potential with the timing of the proton transfer to the ester oxygen, such that diverse aryl and alkyl phosphoesters are turned over with similar catalytic efficiency.

Protein tyrosine phosphatases (PTPs) constitute a large family of signaling enzymes that include both tyrosine specific and dual-specificity phosphatases that hydrolyze pSer/Thr in addition to pTyr. Previous mechanistic studies of PTPs have relied on the highly activated substrate p-nitrophenyl phosphate (pNPP), an aryl phosphate with a leaving group pK(a) of 7. In the study presented here, we employ m-nitrobenzyl phosphate (mNBP), an alkyl phosphate with a leaving group pK(a) of 14.9, which mimics the physiological substrates of the PTPs. We have carried out pH dependence and kinetic isotope effect measurements to characterize the mechanism of two important members of the PTP superfamily: Yersinia PTP (YopH) and Cdc25A. Both YopH and Cdc25A exhibit bell-shaped pH-rate profiles for the hydrolysis of mNBP, consistent with general acid catalysis. The slightly inverse (18)(V/K)(nonbridge) isotope effects (0.9999 for YopH and 0.9983 for Cdc25A) indicate a loose transition state with little nucleophilic participation for both enzymes. The smaller (18)(V/K)(bridge) primary isotope effects (0.9995 for YopH and 1.0012 for Cdc25A) relative to the corresponding isotope effects for pNPP hydrolysis suggest that protonation of the leaving group oxygen at the transition state by the general acid is ahead of P-O bond fission with the alkyl substrate, while general acid catalysis of pNPP by YopH is more synchronous with P-O bond fission. The isotope effect data also confirm findings from previous studies that Cdc25A utilizes general acid catalysis for substrates with a leaving group pK(a) of8, but not for pNPP. Interestingly, the difference in the kinetic isotope effects for the reactions of aryl phosphate pNPP and alkyl phosphate mNBP by the PTPs parallels what is observed in the uncatalyzed reactions of their monoanions. In these reactions, the leaving group is protonated in the transition state, as is the case in PTP-catalyzed reactions. Also, the phosphoryl group in the transition states of the enzymatic reactions does not differ substantially from those of the uncatalyzed reactions. These results provide further evidence that these enzymes do not change the transition state but simply stabilize it.

The reactions of the arylsulfatase A (ASA) from Helix pomatia and that from Aerobacter aerogenes with p-nitrophenyl sulfate were examined by determination of the pH dependence of V m a x /K m and by measurement of kinetic isotope effects. Both enzymes exhibit bell-shaped pH-rate dependences for V m a x /K m . The ASA from Helix pomatia exhibits a more acidic pH optimum (pH 4-5) than the ASA from Aerobacter aerogenes (pH ∼ 7). The sulfuryl transfer from substrate to enzyme is general acid-assisted in both enzymes, but isotope effects indicate differences in the synchronicity of protonation with S-O bond fission. In the reaction of the Helix pomatia enzyme, protonation is synchronous with bond fission and the leaving group is fully neutralized in the transition state. In the reaction catalyzed by the Aerobacter aerogenes ASA, protonation of the leaving group lags behind bond fission and the leaving group bears a partial negative charge in the transition state.

To probe the mechanistic origins of convex Eyring plots that have been observed for alpha-chymotrypsin (alpha-CT)-catalyzed hydrolysis of specific p-nitroanilide substrates [Case, A., and Stein, R. L. (2003) Biochemistry 42, 3335-3348], we determined the temperature-dependence of (15)N-kinetic isotope effects for the alpha-CT-catalyzed hydrolysis of N-succinyl-Phe p-nitroanilide (Suc-Phe-pNA). To provide an interpretational context for these enzymatic isotope effects, we also determined 15N-KIE for alkaline hydrolysis of p-nitroacetanilide. In 0.002 and 2 N hydroxide (30 degrees C), 15N-KIE values are 1.035 and 0.995 (+/-0.001), respectively, and are consistent with the reported [HO-]-dependent change in rate-limiting step from leaving group departure from an anionic tetrahedral intermediate in dilute base, to hydroxide attack in concentrated base. For the alpha-CT-catalyzed hydrolysis of Suc-Phe-pNA, 15N-KIE is on kc/Km and thus reflects structural features of transition states for all reaction steps up to and including acylation of the active site serine. The isotope effect at 35 degrees C is 1.014 (+/-0.001) and suggests that in the transition state for this reaction, departure of leaving group from the tetrahedral intermediate is well advanced. Significantly, 15N-KIE does not vary over the temperature range 5-45 degrees C. This result eliminates one of the competing hypotheses for the convex Eyring plot observed for this reaction, that is, a temperature-dependent change in rate-limiting step within the chemical manifold of acylation, but supports a mechanism in which an isomerization of enzyme conformation is coupled to active site chemistry. We finally suggest that the near absolute temperature-independence of 15N-KIE may point to a unique transition state for this process.

Phosphorothioates, analogues of phosphate esters in which a sulfur replaces an oxygen atom in the phosphoryl group, are competent surrogate substrates for a number of phosphatases. In some cases the thio analogues show similar binding (as estimated by K(m)) while other phosphatases show quite different K(m) values for phosphate compared to phosphorothioate esters. On this basis it was hypothesized that there might be different inhibitory tendencies by the nonhydrolyzable analogues, phosphonothioic acids compared with phosphonic acids. A series of phosphonothioic acids and corresponding phosphonic acids were synthesized and their inhibitory properties were compared toward human placental and E. coli alkaline phosphatases, the protein-tyrosine phosphatase from Yersinia, and the serine/threonine protein phosphatases PP2C and lambda. Sulfur substitution for oxygen gives the phosphonothioic acids pK(a) values that are close to those of phosphate esters, in contrast to the higher pK(a) values typical of phosphonic acids. Despite different steric requirements and differences in charge distribution in the anions of phosphonothioic acids compared with phosphonic acids, it was found that, with some exceptions, differences in inhibitory properties were modest.

Phosphorothioate esters are sometimes used as surrogates for phosphate ester substrates in studies of enzymatic phosphoryl transfer reactions. To gain better understanding of the comparative inherent chemistry of the two types of esters, we have measured equilibrium and kinetic isotope effects for several phosphorothioate esters of p-nitrophenol (pNPPT) and compared the results with data from phosphate esters. The primary (18)O isotope effect at the phenolic group ((18)k(bridge)), the secondary nitrogen-15 isotope effect ((15)k) in the nitro group, and (for the monoester and diester) the secondary oxygen-18 isotope effect ((18)k(nonbridge)) in the phosphoryl oxygens were measured. The equilibrium isotope effect (EIE) (18)k(nonbridge) for the deprotonation of the monoanion of pNPPT is 1.015 +/- 0.002, very similar to values previously reported for phosphate monoesters. The EIEs for complexation of Zn(2+) and Cd(2+) with the dianion pNPPT(2-) were both unity. The mechanism of the aqueous hydrolysis of the monoanion and dianion of pNPPT, the diester ethyl pNPPT, and the triester dimethyl pNPPT was probed using heavy atom kinetic isotope effects. The results were compared with the data reported for analogous phosphate monoester, diester, and triester reactions. The results suggest that leaving group bond fission in the transition state of reactions of the monoester pNPPT is more advanced than for its phosphate counterpart pNPP, while alkaline hydrolysis of the phosphorothioate diester and triester exhibits somewhat less advanced bond fission than that of their phosphate ester counterparts.

A method for the convenient synthesis of phosphonothioates, or phosphonothioic acids, is reported. A significant advantage of the method is the alleviation of the need for purification of intermediates, other than washing with water. No chromatography is needed and the only purification step is the crystallization of the final product. The method uses standard reagents and should be applicable to the synthesis of phosphonothioic acids bearing a range of functional groups.

Cdc25 phosphatases are dual specificity phosphatases that dephosphorylate and activate cyclin-dependent kinases (CDKs), thereby effecting the progression from one phase of the cell cycle to the next. Despite its central role in the cell cycle, relatively little is known about the catalytic mechanism of Cdc25. In order to provide insights into the catalytic mechanism of Cdc25, we have performed a detailed mechanistic analysis of the catalytic domain of human Cdc25A. Our kinetic isotope effect results, Bronsted analysis, and pH dependence studies employing a range of aryl phosphates clearly indicate a dissociative transition state for the Cdc25A reaction that does not involve a general acid for the hydrolysis of substrates with low leaving group pK(a) values (5.45-8.05). Interestingly, our Bronsted analysis and pH dependence studies reveal that Cdc25A employs a different mechanism for the hydrolysis of substrates with high leaving group pK(a) values (8.68-9.99) that appears to require the protonation of glutamic acid 431. Mutation of glutamic acid 431 into glutamine leads to a dramatic drop in the hydrolysis rate for the high leaving group pK(a) substrates and the disappearance of the basic limb of the pH rate profile for the substrate with a leaving group pK(a) of 8.05, indicating that glutamic acid 431 is essential for the efficient hydrolysis of substrates with high leaving group pK(a). We suggest that hydrolysis of the high leaving group pK(a) substrates proceeds through an unfavored but more catalytically active form of Cdc25A, and we propose several models illustrating this. Since the activity of Cdc25A toward small molecule substrates is several orders of magnitude lower than toward the physiological substrate, cyclin-CDK, we suggest that the cyclin-CDK is able to preferentially induce this more catalytically active form of Cdc25A for efficient phosphothreonine and phosphotyrosine dephosphorylation.

Phosphoryl and sulfuryl transfer reactions are essential biological processes. Multiple kinetic isotope effects have provided significant insights into the transition states of these reactions. The data are reviewed for the uncatalyzed reactions of phosphate and sulfate monoesters and for a number of enzymatic phosphoryl transfer reactions. Uncatalyzed phosphoryl and sulfuryl hydrolysis reactions are found to have very similar transition states. The phosphoryl transfer reaction catalyzed by protein-tyrosine phosphatases proceeds by a transition state very similar to that of the uncatalyzed reaction, but isotope effect data reveal an interesting interplay between the conserved arginine and enzyme dynamics involving general acid catalysis.

Although thermodynamically favorable, the uncatalyzed hydrolysis of phosphate monoesters is extraordinarily slow, making phosphatases among the most catalytically efficient enzymes known. Protein-tyrosine phosphatases (PTPs) are ubiquitous in biology, and kinetic isotope effects were one of the key mechanistic tools used to discern molecular details of their catalytic mechanism and the transition state for phosphoryl transfer. Later, the unique level of detail KIEs provided led to deeper questions about the potential role of protein motions in PTP catalysis. The recent discovery that such motions are responsible for different catalytic rates between PTPs arose from questions originating from KIE data showing that the transition states and chemical mechanisms are identical, combined with structural data demonstrating superimposable active sites. KIEs also reveal perturbations to the transition state as mutations are made to residues directly involved in chemistry, and to residues that affect protein motions essential for catalysis.This article is part of a Special Issue entitled: Enzyme Transition States from Theory and Experiment.

Kinetic isotope effects and medium effects have been measured for sulfuryl-transfer reactions of the sulfate ester p-nitrophenyl sulfate (pNPS). The results are compared to those from previous studies of phosphoryl transfer, a reaction with mechanistic similarities. The N-15 and the bridge O-18 isotope effects for the reaction of the pNPS anion are very similar to those of the p-nitrophenyl phosphate (pNPP) dianion. This indicates that in the transition states for both reactions the leaving group bears nearly a full negative charge resulting from a large degree of bond cleavage to the leaving group. The nonbridge O-18 isotope effects support the notion that the sulfuryl group resembles SO(3) in the transition state. The reaction of the neutral pNPS species in acid solution is mechanistically similar to the reaction of the pNPP monoanion. In both cases proton transfer from a nonbridge oxygen atom to the leaving group is largely complete in the transition state. Despite their mechanistic similarities, the phosphoryl- and sulfuryl-transfer reactions differ markedly in their response to medium effects. Increasing proportions of the aprotic solvent DMSO to aqueous solutions of pNPP cause dramatic rate accelerations of up to 6 orders of magnitude, but only a 50-fold rate increase is observed for pNPS. Similarly, phosphoryl transfer from the pNPP dianion to tert-amyl alcohol is 9000-fold faster than the aqueous reaction, while the sulfuryl transfer from the pNPS anion is some 40-fold slower. The enthalpic and entropic contributions to these differing medium effects have been measured and compared.

Dual-specificity phosphatase MKP3 down-regulates mitogenic signaling through dephosphorylation of extracellular regulated kinase (ERK). Unlike a simple substrate-enzyme interaction, the noncatalytic, amino-terminal domain of MKP3 can bind efficiently to ERK, leading to activation of the phosphatase catalytic domain by as much as 100-fold toward exogenous substrates. It has been suggested that ERK activates MKP3 through the stabilization of the active phosphatase conformation, enabling general acid catalysis. Here, we investigated whether Asp-262 of MKP3 is the bona fide general acid and evaluated its contribution to the catalytic steps activated by ERK. Using site-directed mutagenesis, pH rate and Bronsted analyses, kinetic isotope effects, and steady-state and rapid reaction kinetics, Asp-262 was identified as the authentic general acid catalyst, donating a proton to the leaving group oxygen during P-O bond cleavage. Kinetic isotope effects [(18)(V/K)(bridge), (18)(V/K)(nonbridge), and (15)(V/K)] were evaluated for the effect of ERK and of the D262N mutation on the transition state of the phosphoryl transfer reaction. The patterns of the three isotope effects for the reaction with native MKP3 in the presence of ERK are indicative of a reaction where the leaving group is protonated in the transition state, whereas in the D262N mutant, the leaving group departs as the anion. Even without general acid catalysis, the D262N mutant reaction is activated by ERK through increased phosphate affinity ( approximately 8-fold) and the partial stabilization of the transition state for phospho-enzyme intermediate formation ( approximately 4-fold). Based on these analyses, we estimate that dephosphorylation of phosphorylated ERK by the D262N mutant is >1000-fold lower than by native, activated MKP3. Also, the kinetic results suggest that Asp-262 functions as a general base during thiol-phosphate intermediate hydrolysis.

Steady state kinetics and (15)N isotope effects have been used to study the cyclization reaction of uridine 3'-p-nitrophenyl phosphate. The cyclization reaction is catalyzed by transition metal ions and lanthanides, as are substitution reactions of many phosphate esters. Kinetic analysis reveals that the erbium-catalyzed cyclization reaction involves the concerted deprotonation of the 2'-OH group and departure of the leaving group. The transition state is very late, with a very large degree of bond cleavage to the leaving group, which could be due to a large degree of polarization of the Pbond;O bonds by erbium. Copyright 2000 Academic Press.

It has been suggested that the mechanism of alkaline phosphatase (AP) is associative, or triester-like, because phosphorothioate monoesters are hydrolyzed by AP approximately 10(2)-fold slower than phosphate monoesters. This "thio effect" is similar to that observed for the nonenzymatic hydrolysis of phosphate triesters, and is the inverse of that observed for the nonenzymatic hydrolysis of phosphate monoesters. The latter reactions proceed by loose, dissociative transition states, in contrast to reactions of triesters, which have tight, associative transition states. Wild-type alkaline phosphatase catalyzes the hydrolysis of p-nitrophenyl phosphate approximately 70 times faster than p-nitrophenyl phosphorothioate. In contrast, the R166A mutant alkaline phosphatase enzyme, in which the active site arginine at position 166 is replaced with an alanine, hydrolyzes p-nitrophenyl phosphate only about 3 times faster than p-nitrophenyl phosphorothioate. Despite this approximately 23-fold change in the magnitude of the thio effects, the magnitudes of Bronsted beta(lg) for the native AP (-0.77 +/- 0.09) and the R166A mutant (-0.78 +/- 0. 06) are the same. The identical values for the beta(lg) indicate that the transition states are similar for the reactions catalyzed by the wild-type and the R166A mutant enzymes. The fact that a significant change in the thio effect is not accompanied by a change in the beta(lg) indicates that the thio effect is not a reliable reporter for the transition state of the enzymatic phosphoryl transfer reaction. This result has important implications for the interpretation of thio effects in enzymatic reactions.

General acid catalysis in protein tyrosine phosphatases (PTPases) is accomplished by a conserved Asp residue, which is brought into position for catalysis by movement of a flexible loop that occurs upon binding of substrate. With the PTPase from Yersinia, we have examined the effect on general acid catalysis caused by mutations to two conserved residues that are integral to this conformation change. Residue Trp354 is at a hinge of the loop, and Arg409 forms hydrogen bonding and ionic interactions with the phosphoryl group of substrates. Trp354 was mutated to Phe and to Ala, and residue Arg409 was mutated to Lys and to Ala. The four mutant enzymes were studied using steady state kinetics and heavy-atom isotope effects with the substrate p-nitrophenyl phosphate. The data indicate that mutation of the hinge residue Trp354 to Ala completely disables general acid catalysis. In the Phe mutant, general acid catalysis is partially effective, but the proton is only partially transferred in the transition state, in contrast to the native enzyme where proton transfer to the leaving group is virtually complete. Mutation of Arg409 to Lys has a minimal effect on the K(m), while this parameter is increased 30-fold in the Ala mutant. The k(cat) values for R409K and for R409A are about 4 orders of magnitude lower than that for the native enzyme. General acid catalysis is rendered inoperative by the Lys mutation, but partial proton transfer during catalysis still occurs in the Ala mutant. Structural explanations for the differential effects of these mutations on movement of the flexible loop that enables general acid catalysis are presented.

Positive charge is uniformly present in the active sites of all known phosphatases. The postulate that this charge imparts a change to the mechanism and the transition state for phosphoryl transfer was examined by comparing kinetic isotope effects with the substrate p-nitrophenyl phosphate for reactions of the native protein tyrosine phosphatase from Yersinia with data from mutants in which the conserved arginine residue was mutated to Lys or to Ala. The kcat values for both mutants are about 104 less than that of the native enzyme but are still nearly 105-fold faster than the uncatalyzed rate. Steady-state kinetic data as well as isotope effects showed that both mutations interfere with functioning of general acid catalysis. To examine the effect of positive charge on the transition state free of this additional effect, double mutants were made in which general acid catalysis was removed by mutation of Asp356 to either Asn or Ala in addition to the mutation to Arg. The kcat/Km values of D356A and D356N a...

The catalytic reaction of the Mn2+ form of the native bacteriophage λ phosphatase and the H76N mutant was studied with the substrate p-nitrophenyl phosphate using heavy atom isotope effects and pH-...