Impaired Transition State Complementarity in the Hydrolysis of O -Arylphosphorothioates by Protein-Tyrosine Phosphatases

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The mitogen-activated protein kinase phosphatase 3 (MKP3)-catalyzed hydrolysis of aryl phosphates in the absence and presence of extracellular signal-regulated kinase 2 (ERK2) was investigated in order to provide insights into the molecular basis of the ERK2-induced MKP3 activation. In the absence of ERK2, the MKP3-catalyzed hydrolysis of simple aryl phosphates does not display any dependence on pH, viscosity, and the nature of the leaving group. Increased catalytic activity and enhanced affinity for oxyanions are observed for MKP3 in the presence of ERK2. In addition, normal bell-shaped pH dependence on the reaction catalyzed by MKP3 is restored in the presence of ERK2. Collectively, these results suggest that the rate-limiting step in the absence of ERK2 for the MKP3 reaction corresponds to a substrate-induced conformational change in MKP3 involving active site rearrangement and general acid loop closure. The binding of ERK2 to the N-terminal domain of MKP3 facilitates the repositioning of active site residues and speeds up the loop closure in MKP3 such that chemistry becomes rate-limiting in the presence of ERK2. Remarkably, it is found that the extent of ERK2-induced MKP3 activation is substrate dependent, with smaller activation observed for bulkier substrates. Unlike simple aryl phosphates, the MKP3-catalyzed hydrolysis of bulky polycyclic substrates exhibits bell-shaped pH rate profiles in the absence of ERK2. Furthermore, it is found that glycerol can also activate the MKP3-catalyzed reaction, increase the affinity of MKP3 for oxyanion, and restore the bell-shaped pH rate profile for the MKP3-catalyzed reaction. Thus, the rate of repositioning of catalytic groups and the reorienting of the electrostatic environment in the MKP3 active site can be enhanced not only by ERK2 but also by high affinity substrates or by glycerol.A plethora of extracellular stimuli transmit signals into cells through mitogen-activated protein (MAP) 1 kinase cascades. Many mammalian MAP kinase cascades have been characterized (1). The three major MAP kinase cascades include the extracellular signal-regulated protein kinase (ERK) pathway, which responds to stimuli that induce proliferation and differentiation, and the c-Jun N-terminal protein kinase pathway and the p38 kinase pathway, both of which are activated in response to environmental stresses. Each cascade is composed of a three-kinase module: a MAP kinase, a MAP kinase/ERK kinase (MEK) that activates the MAP kinase, and a MEK kinase that activates the MEK. After activation, each MAP kinase phosphorylates a distinct spectrum of substrates, which include key regulatory enzymes, cytoskeletal proteins, nuclear receptors, and several transcription factors.MAP kinase activity is tightly controlled by phosphorylation and dephosphorylation. The activation of the MAP kinase activity requires the dual phosphorylation of the Thr and Tyr residues in the activation loop motif TXY (2-4). Deactivation could occur through the action of serine/threonine protein phosphatases (5), protein-ty...

Section: Fig 4 Effect Of Ph On the Omfp Hydrolysis Reaction Catalyzmentioning

confidence: 99%

Mechanism of Mitogen-activated Protein Kinase Phosphatase-3 Activation by ERK2

Zhou

Zhang

1999

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“…The PTPases utilize the Cys in the signature motif as a nucleophile in the formation of a thiophosphoryl covalent enzyme intermediate (9,10). The invariant Arg residue in the PTPase signature motif functions in substrate binding and in transition state stabilization (11)(12)(13). The initial phosphoryl transfer from the substrate to the enzyme is assisted by a conserved Asp on a surface loop, which protonates the leaving group (14,15), thereby acting as a general acid catalyst.…”

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confidence: 99%

The Mechanism of Dephosphorylation of Extracellular Signal-regulated Kinase 2 by Mitogen-activated Protein Kinase Phosphatase 3

Zhao¹,

Zhang²

2001

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167

156

The mitogen-activated protein (MAP) kinase phosphatase-3 (MKP3) is a dual specificity phosphatase that specifically inactivates one subfamily of MAP kinases, the extracellular signal-regulated kinases (ERKs). Inactivation of MAP kinases occurs by dephosphorylation of Thr(P) and Tyr(P) in the TXY kinase activation motif. To gain insight into the mechanism of ERK2 inactivation by MKP3, we have carried out an analysis of the MKP3-catalyzed dephosphorylation of the phosphorylated ERK2. We find that ERK2/pTpY dephosphorylation by MKP3 involves an ordered, distributive mechanism in which MKP3 binds the bisphosphorylated ERK2/pTpY, dephosphorylates Tyr(P) first, dissociates and releases the monophosphorylated ERK2/pT, which is then subjected to dephosphorylation by a second MKP3, yielding the fully dephosphorylated ERK2. The bisphosphorylated ERK2 is a highly specific substrate for MKP3 with a k cat /K m of 3.8 ؋ 10 6 M ؊1 s ؊1 , which is more than 6 orders of magnitude higher than that for small molecule aryl phosphates and an ERK2-derived phosphopeptide encompassing the pTEpY motif. This strikingly high substrate specificity displayed by MKP3 may result from a combination of high affinity binding interactions between the N-terminal domain of MKP3 and ERK2 and specific ERK2-induced allosteric activation of the MKP3 C-terminal phosphatase domain.

“…It has been shown that the cysteine residue in this motif functions as a nucleophile, attacking the substrate phosphate atom directly and displacing the leaving group to form a phosphocysteine intermediate (17)(18)(19)(20)(21)(22). The arginine in this motif has been shown to be particularly important for transition state stabilization (23)(24)(25) and is essential for catalysis (26 -28). For Cdc25B, the cysteine and arginine in the PTPase signature motif have been shown by site-directed mutagenesis to be required for catalysis (29).…”

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confidence: 99%

The Catalytic Mechanism of Cdc25A Phosphatase

McCain

Catrina

Hengge

et al. 2002

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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.