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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

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).

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.

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.

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.

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 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.

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.

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.

© 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.

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.

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.

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.

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.

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.

Activation of calcineurin by Mn2+ and Mg2+ was compared using a heavy atom isotope analogue of the substrate p-nitrophenyl phosphate (pNPP). Heavy atom isotope effects were measured for Mg2+ activation and compared to published results of the isotope effects with Mn2+ as the activating metal. Isotope effects were measured for the kinetic parameter Vmax/Km at the nonbridging oxygen atoms [18(V/K)nonbridge]; at the position of bond cleavage in the bridging oxygen atom [18(V/K)bridge]; and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The isotope effects increased in magnitude upon changing from an optimal pH to a nonoptimal pH; the 18(V/K)bridge effect increased from 1.0154 (+/-0.0007) to 1.0198 (+/-0.0002), and the 15(V/K) effect increased from 1.0018 (+/-0. 0002) to 1.0021 (+/-0.0003). The value for 18(V/K)nonbridge is 0. 9910 (+/-0.0003) at pH 7.0. As with Mn2+, the 18(V/K)nonbridge isotope effect indicated that the dianion was the substrate for catalysis, and that a dissociative transition state was operative for the phosphoryl transfer. Comparison to results for Mn2+ activation suggested that chemistry was more rate-limiting with Mg2+ than with Mn2+. Changing the activating metal concentration showed opposite trends with increasing Mg2+ increasing the commitment factor and seemingly making the chemistry less rate-limiting. The influence of viscosity was evaluated as well to gauge the role of chemistry. The activation of calcineurin-catalyzed hydrolysis of pNPP1 by Mg2+ or Mn2+ at pH 7.0 was compared in the presence of viscogens, glycerol and poly(ethylene glycol). Increasing glycerol caused different effects with the two activators. With Mn2+ as the activator, calcineurin activity showed a normal response with kcat and kcat/Km decreasing with viscosity. There was an inverse response with Mg2+ as the activator as values of kcat/Km increased with viscosity. From values of the normalized kcat/Km with Mn2+, the chemistry was found to be partially rate-limiting, consistent with previous heavy atom isotope studies (22). The effect observed for Mg2+ seems consistent with a change in the rate-limiting step for the two different metals at pH 7.0.

Isotope effects have been measured for the reaction of the human dual-specific phosphatase VHR with p-nitrophenyl phosphate (pNPP). Isotope effects in the nonbridge oxygen atoms, in the bridge oxygen atom, and in the nitrogen atom were measured by the competitive method using an isotope ratio mass spectrometer. These are isotope effects on V/K, and give information on the chemical step of phosphoryl transfer from substrate to the enzymatic nucleophile Cys-124. With native VHR, 18(V/K)nonbridge = 1.0003 +/- 0.0003, 18(V/K)bridge = 1.0118 +/- 0.0020, and 15(V/K) = 0.9999 +/- 0.0004. The values are similar to the intrinsic isotope effects for the uncatalyzed reaction, indicating that the chemical step is rate-limiting with the pNPP substrate. The transition-state structure resembles that for the uncatalyzed reaction and those previously found for the protein-tyrosine phosphatases YOP51 and PTP1, and is highly dissociative with P-O bond cleavage and protonation of the leaving group by the general acid Asp-92 both well advanced. The D92N mutant exhibits a transition state similar to that of the uncatalyzed reaction of the pNPP dianion, dissociative and with the leaving group departing as the nitrophenolate anion. The S131A mutation causes an increase in the pKa of the nucleophilic Cys, but the isotope effect data are unchanged from those for the native enzyme, indicating no effects of this increase in nucleophilicity on transition-state structure. The double mutant D92N/S131A manifests both the increase in pKa of the nucleophilic Cys and the loss of general acid assistance to the leaving group. In the absence of the general acid, the change in nucleophile pKa results in an increase in 18(V/K)nonbridge from 1.0019 (with D92N) to 1.0031 (with D92N/S131A), indicating loss of P-O nonbridge bond order in the transition state. It is concluded that this is more likely caused by electrostatic effects rather than resulting from increased nucleophile-phosphorus bonding in a less dissociative transition state, although the latter explanation cannot be excluded on the basis of the present data. Electrostatic effects between the thiolate anion nucleophile and the phosphoryl group may be an important part of the driving force for catalysis in this family of enzymes.

Phosphorylation reactions catalyzed by kinases and phosphatases play an indispensible role in cellular signaling, and their malfunctioning is implicated in many diseases. A better understanding of the catalytic mechanism will help design novel and effective mechanism-based inhibitors of these enzymes. In this work, ab initio quantum mechanical/molecular mechanical studies are reported for the phosphoryl transfer reaction catalyzed by a cyclin-dependent kinase, CDK2. Our results suggest that an active-site Asp residue, rather than ATP as previously proposed, serves as the general base to activate the Ser nucleophile. The corresponding transition state features a dissociative, metaphosphate-like structure, stabilized by the Mg(2+) ion and several hydrogen bonds. The calculated free-energy barrier is consistent with experimental values. Implications of our results in this and other protein kinases are discussed.

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 Fe(3+)-Fe(2+) pig PAP (uteroferrin; Uf) and its Fe(3+)-Mn(2+) derivative to investigate the effect of metal ion substitution on the mechanism of catalysis. Replacement of the Fe(2+) by Mn(2+) 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 Fe(3+) site. These data also indicate that in both metal forms of Uf, phenyl phosphate hydrolysis occurs faster than formation of a mu-1,3 phosphate complex. The slower rate of interaction between substrate and the Fe(3+) 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 Fe(3+). The faster rates of interaction with the Fe(3+) site in the Fe(3+)-Mn(2+) derivative than the native Fe(3+)-Fe(2+) 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 ([Co(2)(tacn)(2)(OH)(2){O(2)P(Me)OAr}](3+); tacn = 1,4,7-triazacyclononane; substituent = m-F, p-NO(2) (1a); p-NO(2) (1b); m-NO(2) (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 beta(lg) 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 (18)k(lg) = 1.0228 and (15)k = 1.0014, at the nonbridging phosphoryl oxygens (18)k(nonbridge) = 0.9954, and at the nucleophilic oxygen(18)k(nuc) = 1.0105. The KIEs and the beta(lg) 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.

DNA ligase seals nicks in dsDNA using chemical energy of the phosphoanhydride bond in ATP or NAD(+) and assistance of a divalent metal cofactor Mg(2+). Molecular details of ligase catalysis are essential for understanding the mechanism of metal-promoted phosphoryl transfer reactions in the living cell responsible for a wide range of processes, e.g., DNA replication and transcription, signaling and differentiation, energy coupling and metabolism. Here we report a single-turnover (31)P solid-state NMR study of adenylyl transfer catalyzed by DNA ligase from bacteriophage T4. Formation of a high-energy covalent ligase-nucleotide complex is triggered in situ by the photo release of caged Mg(2+), and sequentially formed intermediates are monitored by NMR. Analyses of reaction kinetics and chemical-shift changes indicate that the pentacoordinated phosphorane intermediate builds up to 35% of the total reacting species after 4-5 h of reaction. This is direct experimental evidence of the associative nature of adenylyl transfer catalyzed by DNA ligase. NMR spectroscopy in rotating solids is introduced as an analytical tool for recording molecular movies of reaction processes. Presented work pioneers a promising direction in structural studies of biochemical transformations.

The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a promiscuous binuclear metallohydrolase that catalyzes the hydrolysis of mono-, di-, and triester substrates, including some organophosphate pesticides and products of the degradation of nerve agents. GpdQ has attracted recent attention as a promising enzymatic bioremediator. Here, we have investigated the catalytic mechanism of this versatile enzyme using a range of techniques. An improved crystal structure (1.9 A resolution) illustrates the presence of (i) an extended hydrogen bond network in the active site, and (ii) two possible nucleophiles, i.e., water/hydroxide ligands, coordinated to one or both metal ions. While it is at present not possible to unambiguously distinguish between these two possibilities, a reaction mechanism is proposed whereby the terminally bound H2O/OH(-) acts as the nucleophile, activated via hydrogen bonding by the bridging water molecule. Furthermore, the presence of substrate promotes the formation of a catalytically competent binuclear center by significantly enhancing the binding affinity of one of the metal ions in the active site. Asn80 appears to display coordination flexibility that may modulate enzyme activity. Kinetic data suggest that the rate-limiting step occurs after hydrolysis, i.e., the release of the phosphate moiety and the concomitant dissociation of one of the metal ions and/or associated conformational changes. Thus, it is proposed that GpdQ employs an intricate regulatory mechanism for catalysis, where coordination flexibility in one of the two metal binding sites is essential for optimal activity.

The purple acid phosphatases (PAPs) are bimetallophosphatases that are reported to lack phosphodiesterase activity based on the absence of reactivity with bis(p-nitrophenyl) phosphate. Yet, model systems designed to mimic the PAP active site preferentially hydrolyze phosphodiesters. We report that the PAPs from pig and from red kidney bean efficiently hydrolyze the diesters methyl nitrophenyl phosphate and ethyl nitrophenyl phosphate. The details of the diester reaction give insights into the sequential steps involved in binding and hydrolysis by the metal center. Methyl phosphate monoester, the initial product of the reaction with methyl nitrophenyl phosphate, is subsequently hydrolyzed without release into solution. The turnover number for this reaction is faster than that for the reaction of free methyl phosphate. This is consistent with a mechanism for monoester hydrolysis consisting of initial coordination to the divalent metal, followed by slower partially rate-limiting displacement of hydroxide from Fe(III) to give the catalytically active bidentately bound substrate. The reaction with the diester substrate is proposed to proceed by coordination to the divalent metal ion followed by nucleophilic attack by the Fe(III)-coordinated hydroxide to give a bidentately bound monoester poised for attack by the bridging mu-hydroxide. This mechanism eliminates the need for the second, slow binding event in monoester hydrolysis.

The catalytic promiscuity of E. coli alkaline phosphatase (AP) and many other enzymes provides a unique opportunity to dissect the origin of enzymatic rate enhancements via a comparative approach. Here, we use kinetic isotope effects (KIEs) to explore the origin of the 109-fold greater catalytic proficiency by AP for phosphate monoester hydrolysis relative to sulfate monoester hydrolysis. The primary 18O KIEs for the leaving group oxygen atoms in the AP-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) and p-nitrophenylsulfate (pNPS) decrease relative to the values observed for nonenzymatic hydrolysis reactions. Prior linear free energy relationship results suggest that the transition states for AP-catalyzed reactions of phosphate and sulfate esters are "loose" and indistinguishable from that in solution, suggesting that the decreased primary KIEs do not reflect a change in the nature of the transition state but rather a strong interaction of the leaving group oxygen atom with an active site Zn2+ ion. Furthermore, the primary KIEs for the two reactions are identical within error, suggesting that the differential catalysis of these reactions cannot be attributed to differential stabilization of the leaving group. In contrast, AP perturbs the KIE for the nonbridging oxygen atoms in the reaction of pNPP but not pNPS, suggesting a differential interaction with the transferred group in the transition state. These and prior results are consistent with a strong electrostatic interaction between the active site bimetallo Zn2+ cluster and one of the nonbridging oxygen atoms on the transferred group. We suggest that the lower charge density of this oxygen atom on a transferred sulfuryl group accounts for a large fraction of the decreased stabilization of the transition state for its reaction relative to phosphoryl transfer.

Evidence indicates that phosphate monoesters undergo hydrolysis by a loose transition state with extensive bond fission to the leaving group. It has been proposed that part of the high dependence of the rate on the leaving group pKa (betalg = -1.23) arises from weaker ester bonds in the reactants as the pKa of the leaving group decreases, on the basis of X-ray structures and calculations. In contrast, IR and Raman studies suggest that the leaving group has little effect on the length of the P-OR bridging bond in solution. To gather additional data on this issue, we have used (18)O isotopic shifts in 31P NMR to monitor the bond order of P-O bonds in a range of phosphate esters with different leaving groups. Using this technique, we have been able to evaluate whether significant changes are observed in the P-O bond orders for the bridging and nonbridging positions of methyl, ethyl, phenethyl, propargyl, phenyl, and p-nitrophenyl phosphate using [(16)O(18)O] labeled species in deuterium oxide. The results indicate that the bridging and nonbridging bond orders to phosphorus in phosphate monoesters are not significantly altered by differences in the pKa of the leaving group or by the counterion of the phosphate ester dianion.

Earlier work on the hydrolysis of aryl phosphinothioate esters has led to contradictory mechanistic conclusions. To resolve this mechanistic ambiguity, we have measured linear free energy relationships (beta(nuc) and beta(lg)) and kinetic isotope effects for the reactions of oxyanions with aryl dimethylphosphinothioates. For the attack of nucleophiles on 4-nitrophenyl dimethylphosphinothioate, beta(nuc) = 0.47 +/- 0.05 for phenoxide nucleophiles (pK(a)11) and beta(nuc) = 0.08 +/- 0.01 for hydroxide and alkoxide nucleophiles (pK(a)or= 11). Linearity of the plot in the range that straddles the pK(a) of the leaving group (4-nitrophenoxide, pK(a) 7.14) is indicative of a concerted mechanism. The much lower value of beta(nuc) for the more basic nucleophiles reveals the importance of a desolvation step prior to rate-limiting nucleophilic attack. The reactions of a series of substituted aryl dimethylphosphinothioate esters give the same value of beta(lg) with the nucleophiles HO(-) (beta= -0.54 +/- 0.03) and PhO(-) (beta = -0.52 +/- 0.09). A significantly better Hammett correlation is obtained with sigma(-) than with sigma or sigma degrees , as expected for a transition state involving rate-limiting cleavage of the P-OAr bond. The (18)O KIE at the position of bond fission ((18)k = 1.0124 +/- 0.0008) indicates the P-O bond is approximately 40% broken, and the (15)N KIE in the leaving group ((15)k = 1.0009 +/- 0.0003) reveals the nucleofuge carries about a third of a negative charge in the transition state. Thus, both the LFER and KIE data are consistent with a concerted reaction and disfavor a stepwise mechanism.

Kinetic isotope effects in the nucleophile and leaving group were obtained for the reaction of p-nitrophenyl phosphate monoester coordinated to a dinuclear Co(III) complex. The metal complex of the p-nitrophenyl phosphate monoester was found to hydrolyze by a single-step concerted mechanism, with significant nucleophilic participation in the transition state. By contrast, the hydrolysis of uncomplexed p-nitrophenyl phosphate occurs by a very loose transition state with little bond formation to the nucleophile. Previously, the metal complex of the diester methyl-p-nitrophenyl phosphate was found to hydrolyze via a two-step addition−elimination mechanism, in contrast to the concerted hydrolysis mechanism followed by uncomplexed diesters with the p-nitrophenolate leaving group. These results show that coordination to a metal complex can significantly alter the mechanism of phosphoryl transfer.

15 N isotope effects in the nitro group and 18 O isotope effects in the phenolic oxygen have been measured for the hydrolysis of ethyl p -nitrophenyl phosphate catalyzed by several metal ions. Co(III)–cyclen at pH 7, 50 °C, gave an 15 N isotope effect of 0.12% and an 18 O one of 2.23%, showing that P–O cleavage is rate limiting and the bond is ∼50% broken in the transition state. The active catalyst is a dimer and the substrate is presumably coordinated to the open site of one Co(III), and is attacked by hydroxide coordinated to the other Co(III). Co(III)–tacn under the same conditions shows a similar 15 N isotope effect (0.13%), but a smaller 18 O one (0.8%). Zn(II)–cyclen at pH 8.5, 80 °C, gave an 15 N isotope effect of 0.05% and an 18 O one of 0.95%, suggesting an earlier transition state. The catalyst in this case is monomeric, and thus the substrate is coordinated to one position and attacked by a cis -coordinated hydroxide. Eu(III) at pH 6.5, 50 °C, shows a very large 15 N isotope effect of 0.34% and a 1.6% 18 O isotope effect. The large 15 N isotope effect argues for a late transition state or Eu(III) interaction with the nitro group, and was also seen in Eu(III)-catalyzed hydrolysis of p -nitrophenyl phosphate [Bioorg. Chem. 28 (2000) 283].

Isotope effects in the nucleophile and in the leaving group were measured to gain information about the mechanism and transition state of the hydrolysis of methyl p-nitrophenyl phosphate complexed to a dinuclear cobalt complex. The complexed diester undergoes hydrolysis about 1011 times faster than the corresponding uncomplexed diester. The kinetic isotope effects indicate that this rate acceleration is accompanied by a change in mechanism. A large inverse 18O isotope effect in the bridging hydroxide nucleophile (0.937 +/- 0.002) suggests that nucleophilic attack occurs before the rate-determining step. Large isotope effects in the nitrophenyl leaving group (18Olg = 1.029 +/- 0.002, 15N = 1.0026 +/- 0.0002) indicate significant fission of the P-O ester bond in the transition state of the rate-determining step. The data indicate that in contrast to uncomplexed diesters, which undergo hydrolysis by a concerted mechanism, the reaction of the complexed diester likely proceeds via an addition-elimination mechanism. The rate-limiting step is expulsion of the p-nitrophenyl leaving group from the intermediate, which proceeds by a late transition state with extensive bond fission to the leaving group. This represents a substantial change in mechanism from the hydrolysis of uncomplexed aryl phosphate diesters.

Previous work by Kirby and co-workers revealed a significant acceleration of the rate of hydrolysis of p-nitrophenyl phosphate by added dipolar solvents such as DMSO. Activation parameters and kinetic isotope effects have been measured to ascertain the origin of this effect. The generality of this phenomenon was examined with a series of esters with more basic leaving groups. Computational analyses of the effects of desolvation of dianionic phosphate monoesters were carried out, and the possible effect of the transfer from water to the active site of alkaline phosphatase was modeled. The results are consistent with a desolvation-induced weakening of the P-O ester bond in the ground state. Other aryl phosphate esters show similar rate accelerations at high fractions of DMSO, but phenyl and methyl phosphates do not, and their hydrolysis reactions are actually slowed by these conditions.

In order to evaluate various mechanistic proposals that have been made regarding the mechanism of the first step of the reaction catalyzed by protein-tyrosine phosphatases, new experimental data have been obtained, and some existing data have been carefully reevaluated. New kinetic isotope effect data for the uncatalyzed hydrolysis of p-nitrophenyl phosphate allow a better evaluation of previously reported data from enzymatic reactions with this substrate. The interpretation, and misinterpretation, of pH rate studies is considered. The pathway of this reaction has been modeled computationally and is found to be generally consistent with experimental studies, except for the extent of proton transfer to the leaving group.

The reactions of p-nitrophenyl phosphate (pNPP) with the low-molecular mass tyrosine phosphatase Stp1 and with the mutants D128N, D128A, D128E, and S18A have been studied by measurement of heavy-atom isotope effects in the substrate. The isotope effects were measured at the nonbridging oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom (the site of bond cleavage) [18(V/K)bridge], and at the nitrogen atom in the nitrophenol leaving group [15(V/K)]. The results with native Stp1 were 1.0160 +/- 0.0005 for 18(V/K)bridge, 1.0007 +/- 0.0001 for 15(V/K), and 1.0018 +/- 0.0003 for 18(V/K)nonbridge. The values for 18(V/K)nonbridge and 15(V/K) differ from those previously measured with other protein-tyrosine phosphatases and from those of the aqueous hydrolysis reaction of pNPP. The values indicate that in the transition state of the native Stp1 reaction the leaving group bears a partial negative charge, and there is nucleophilic interaction between the Cys nucleophile, and the phosphoryl group, causing some decrease in the nonbridge P-O bond order. The transition state remains highly dissociative with respect to the degree of bond cleavage to the leaving group. Mutation of the general acid from aspartic acid to glutamic acid slows catalysis but causes no change in the isotope effects and thus does not alter the degree of proton transfer to the leaving group in the transition state. Mutations of this residue to asparagine or alanine give values for 18(V/K)bridge of about 1.029, for 15(V/K) of about 1.003, and for 18(V/K)nonbridge of 1.0010 (D128A) to 1.0024 (D128N). These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion and indicate more nucleophilic participation than in the aqueous hydrolysis of the pNPP dianion, just as in the native enzyme. The isotope effects with the S18A mutant, in which a hydrogen bonding stabilization of the anionic Cys nucleophile has been removed, were within experimental error of those with the native enzyme, indicating that this alteration has no effect on the transition state for phosphoryl transfer from pNPP.

{sup 18}O isotope effects in and {sup 18}O labelling of nitrophenols is of interest. The authors found and here report that ortho- and paranitrophenols display facile phenolic oxygen exchange in basic solution. Kinetics of these exchange reactions were studied and results of exchange rates are reported. 8 refs., 2 figs., 1 tab.

Protein-tyrosine phosphatases and serine/threonine protein phosphatases utilize very different catalytic machinery to catalyze phosphoryl transfer reactions. Tyrosine is a better leaving group than serine or threonine, having a pKa more than three units lower. Has the difference in the catalytic machinery used by these enzyme families evolved as a result of the difference in the lability of their substrates? Are the transition states for phosphoryl transfer similar for the two classes of enzymes? This review summarizes what has been learned from kinetic isotope effects about the nature of enzymatic phosphoryl transfer, and how the enzymatic mechanisms compare to uncatalyzed phosphoryl transfer reactions.