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.
Many studies have implicated a role for conformational motions during the catalytic cycle, acting to optimize the binding pocket or facilitate product release, but a more intimate role in the chemical reaction has not been described. We address this by monitoring active-site loop motion in two protein tyrosine phosphatases (PTPs) using NMR spectroscopy. The PTPs, YopH and PTP1B, have very different catalytic rates, however we find in both that the active-site loop closes to its catalytically competent position at rates that mirror the phosphotyrosine cleavage kinetics. This loop contains the catalytic acid, suggesting that loop closure occurs concomitantly with the protonation of the leaving group tyrosine and explains the different kinetics of two otherwise chemically and mechanistically indistinguishable enzymes.
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 Å 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 dephosphorylation of p-nitrophenyl phosphate by Yersinia protein-tyrosine phosphatase (PTPase) and by the rat PTP1 has been examined by measurement of heavy-atom isotope effects at the nonbridge oxygen atoms [18(V/K)nonbridge], at the bridging oxygen atom [18(V/K)bridge], and the nitrogen atom in the leaving group 15(V/K). The effects were measured using an isotope ratio mass spectrometer by the competitive method and thus are effects on V/K. The results for the Yersinia PTPase and rat PTP1, respectively, are 1.0142 +/- 0.0004 and 1.0152 +/- 0.0006 for 18(V/K)bridge; 0.9981 +/- 0.0015 and 0.9998 +/- 0.0013 for 18(V/K)nonbridge; and 1.0001 +/- 0.0002 and 0.9999 +/- 0.0003 for 15(V/K). The magnitudes of the isotope effects are similar to the intrinsic values measured in solution, indicating that the chemical step is rate-limiting for V/K. The transition state for phosphorylation of the enzyme is dissociative in character, as is the case in solution. Binding of the substrate is rapid and reversible, as is the binding-induced conformational change which brings the catalytic general acid into the active site. Cleavage of the P-O bond and proton transfer from the general acid Asp to the leaving group are both far advanced in the transition state, and there is no development of negative charge on the departing leaving group. Experiments with several general acid mutants give values for 18(V/K)bridge of around 1.0280, 15(V/K) of about 1.002, and 18(V/K)nonbridge effects of from 1.0007 to 1.0022. These data indicate a dissociative transition state with the leaving group departing as the nitrophenolate anion but suggest more nucleophilic participation than in the solution reaction.
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 phosphorylation of tyrosine residues by protein-tyrosine kinases and the reverse action by protein-tyrosine phosphatases (PTPs) 4 is a common mechanism for the control of biological pathways (1-3). Protein-tyrosine phosphatase 1B (PTP1B) is a biomedically important phosphatase with several roles, including negative regulation of insulin signaling by dephosphorylation of the insulin receptor tyrosine kinase (4). Knock-out studies show that loss of PTP1B is associated with an increased insulin sensitivity and suppression of weight gain in mice (5). As a result, this enzyme has been considered a significant target for treatment of type 2 diabetes and obesity (6, 7). PTP1B also down-regulates cell growth by dephosphorylating the epidermal growth factor receptor (8). Overexpression has been observed in human breast and ovarian cancer, where it is believed to suppress potential tumors by antagonizing signaling of oncogenic factors (9, 10). Other PTPs are known virulence factors for a number of human diseases (11-13).Reactions catalyzed by PTPs take place by a ping-pong mechanism ( Fig. 1) (2). In the first step, a nucleophilic cysteine thiolate attacks the phosphate ester moiety of the substrate, resulting in formation of a phosphoenzyme intermediate with release of the peptidyl tyrosine. The second step occurs via attack of water on the phosphoenzyme intermediate and yields the final products inorganic phosphate and the regenerated enzyme. The central binding site for the substrate is the P-loop, a region at the bottom of a pocket that includes the nucleophilic cysteine and backbone amide groups oriented in a horseshoe fashion. The amide protons in the P-loop, together with a conserved arginine residue, hydrogen-bond to the phosphoryl group of the substrate and orient it for nucleophilic attack, providing transition state (TS) stabilization. Substrate binding is followed by conformational changes that culminate with closure of the activ...
Acyl and phosphoryl transfer are important biochemical reactions. We have been using isotope effects caused by O-18, N-15, C-13, and deuterium substitution to examine the mechanisms and transition-state structures for enzymatic and nonenzymatic transfers of phosphoryl and acyl groups. Phosphoryl transfers from phosphate monoesters are highly dissociative, although not truly stepwise in protic solvents or in enzymatic reactions. Phosphodiesters show ANDN (SN2) reactions, whereas triester hydrolyses involve more associative transition states. Except under acidic conditions, true phosphorane intermediates likely form only when geometry requires (i.e., when the leaving group cannot be axial until pseudorotation of the phosphorane). Enzymatic phosphoryl transfers appear similar to nonenzymatic ones. The reactions of oxygen or sulfur nucleophiles with p-nitrophenyl acetate are concerted with a tetrahedral transition state, which is more dissociative with sulfur than with oxygen. Enzymatic hydrolyses of p-nitrophenyl acetate are also concerted reactions.
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