Negative feedback predominates over cross-regulation to control ERK MAPK activity in response to FGF signalling in embryos

Expression of the gene encoding the MKP-3/Pyst1 protein phosphatase, which inactivates ERK MAPK, is induced by FGF. However, which intracellular signalling pathway mediates this expression is unclear, with essential roles proposed for both ERK and PI(3)K in chick embryonic limb. Here, we report that MKP-3/Pyst1 expression is sensitive to inhibition of ERK or MAPKK, that endogenous MKP-3/Pyst1 co-localizes with activated ERK, and expression of MKP-3/Pyst1 in mice lacking PDK1, an essential mediator of PI(3)K signalling. We conclude that MKP-3/Pyst1 expression is mediated by ERK activation and that negative feedback control predominates in limiting the extent of FGF-induced ERK activity.     

The hepatitis E virus open reading frame 3 protein activates ERK through binding and inhibition of the MAPK phosphatase

The hepatitis E virus causes acute viral hepatitis endemic in much of the developing world and is a serious public health problem. However, due to the lack of an in vitro culture system or a small animal model, its biology and pathogenesis are poorly understood. We have shown earlier that the ORF3 protein (pORF3) of hepatitis E virus activates ERK, a member of the MAPK superfamily. Here we have explored the mechanism of pORF3-mediated ERK activation and demonstrated it to be independent of the Raf/MEK pathway. Using biochemical assays, yeast two-hybrid analysis, and intracellular fluorescence resonance energy transfer we showed that pORF3 binds Pyst1, a prototypic member of the ERK-specific MAPK phosphatase. The binding regions in the two proteins were mapped to the N terminus of pORF3 and a central portion of Pyst1. Expression of pORF3 protected ERK from the inhibitory effects of ectopically expressed Pyst1. This is the first example of a viral protein regulating ERK activation by inhibition of its cognate dual specificity phosphatase.     

Role of two active site Glu residues in the molecular action of botulinum neurotoxin endopeptidase

Botulinum neurotoxin type A (BoNT/A) light chain (LC) is a zinc endopeptidase that causes neuroparalysis by blocking neurotransmitter release at the neuromuscular junctions. The X-ray crystal structure of the toxin reveals that His223 and His227 of the Zn(2+) binding motif HEXXH directly coordinate the active site zinc. Two Glu residues (Glu224 and Glu262) are also part of the active site, with Glu224 coordinating the zinc via a water molecule whereas Glu262 coordinates the zinc directly as the fourth ligand. In the past we have investigated the topographical role of Glu224 by replacing it with Asp thus reducing the side chain length by 1.4 A that reduced the endopeptidase activity dramatically [L. Li, T. Binz, H. Niemann, and B.R. Singh, Probing the role of glutamate residue in the zinc-binding motif of type A botulinum neurotoxin light chain, Biochemistry 39 (2000) 2399-2405]. In this study we have moved the Glu 224 laterally by a residue (HXEXH) to assess its positional influence on the endopeptidase activity, which was completely lost. The functional implication of Glu262 was investigated by replacing this residue with aspartate and glutamine using site-directed mutagenesis. Substitution of Glu262 with Asp resulted in a 3-fold decrease in catalytic efficiency. This mutation did not induce any significant structural alterations in the active site and did not interfere with substrate binding. Substitution of Glu262 with Gln however, dramatically impaired the enzymatic activity and this is accompanied by global alterations in the active site conformation in terms of topography of aromatic amino acid residues, zinc binding, and substrate binding, resulting from the weakened interaction between the active site zinc and Gln. These results suggest a pivotal role of the negatively charged carboxyl group of Glu262 which may play a critical role in enhancing the stability of the active site with strong interaction with zinc. The zinc may thus play structural role in addition to its catalytic role.     

Negative feedback regulation of FGF signaling levels by Pyst1/MKP3 in chick embryos

BACKGROUND: The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. RESULTS: We have cloned chicken Pyst1/Mkp3 and show that high-level expression in neural plate correlates with active MAPK. We show that FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). We further show by applying a specific MAP kinase kinase inhibitor (PD184352) that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. CONCLUSIONS: Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. Our results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates.     

Structure and mechanism of the 2′,3′ phosphatase component of the bacterial Pnkp-Hen1 RNA repair system

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5′ kinase, a central 2′,3′ phosphatase and a C-terminal ligase. The phosphatase module is a Mn²⁺-dependent phosphodiesterase–monoesterase that dephosphorylates 2′,3′-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn²⁺ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage λ phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn²⁺ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.     

Residue 259 in protein-tyrosine phosphatase PTP1B and PTPalpha determines the flexibility of glutamine 262

To study the flexibility of the substrate-binding site and in particular of Gln262, we have performed adiabatic conformational search and molecular dynamics simulations on the crystal structure of the catalytic domain of wild-type protein-tyrosine phosphatase (PTP) 1B, a mutant PTP1B(R47V,D48N,M258C,G259Q), and a model of the catalytically active form of PTPalpha. For each molecule two cases were modeled: the Michaelis-Menten complex with the substrate analogue p-nitrophenyl phosphate (p-PNPP) bound to the active site and the cysteine-phosphor complex, each corresponding to the first and second step of the phosphate hydrolysis. Analyses of the trajectories revealed that in the cysteine-phosphor complex of PTP1B, Gln262 oscillates freely between the bound phosphate group and Gly259 frequently forming, as observed in the crystal structure, a hydrogen bond with the backbone oxygen of Gly259. In contrast, the movement of Gln262 is restricted in PTPalpha and the mutant due to interactions with Gln259 reducing the frequency of the oscillation of Gln262 and thereby delaying the positioning of this residue for the second step in the catalysis, as reflected experimentally by a reduction in k(cat). Additionally, in the simulation with the Michaelis-Menten complexes, we found that a glutamine in position 259 induces steric hindrance by pushing the Gln262 side chain further toward the substrate and thereby negatively affecting K(m) as indicated by kinetic studies. Detailed analysis of the water structure around Gln262 and the active site Cys215 reveals that the probability of finding a water molecule correctly positioned for catalysis is much larger in PTP1B than in PTP1B(R47V,D48N,M258C,G259Q) and PTPalpha, in accordance with experiments.     

How calcium inhibits the magnesium-dependent enzyme human phosphoserine phosphatase.

The structure of the Mg(2+)-dependent enzyme human phosphoserine phosphatase (HPSP) was exploited to examine the structural and functional role of the divalent cation in the active site of phosphatases. Most interesting is the biochemical observation that a Ca(2+) ion inhibits the activity of HPSP, even in the presence of added Mg(2+). The sixfold coordinated Mg(2+) ion present in the active site of HPSP under normal physiological conditions, was replaced by a Ca(2+) ion by using a crystallization condition with high concentration of CaCl(2) (0.7 m). The resulting HPSP structure now shows a sevenfold coordinated Ca(2+) ion in the active site that might explain the inhibitory effect of Ca(2+) on the enzyme. Indeed, the Ca(2+) ion in the active site captures both side-chain oxygen atoms of the catalytic Asp20 as a ligand, while a Mg(2+) ion ligates only one oxygen atom of this Asp residue. The bidentate character of Asp20 towards Ca(2+) hampers the nucleophilic attack of one of the Asp20 side chain oxygen atoms on the phosphorus atom of the substrate phosphoserine.     

Effects of protonation state of Asp181 and position of active site water molecules on the conformation of PTP1B

In protein tyrosine phosphatase 1B (PTP1B), the flexible WPD loop adopts a closed conformation (WPD_closed) in the active state of PTP1B, bringing the catalytic Asp181 close to the active site pocket, while WPD loop is in an open conformation (WPD_open) in the inactive state. Previous studies showed that Asp181 may be protonated at physiological pH, and ordered water molecules exist in the active site. In the current study, molecular dynamics simulations are employed at different Asp181 protonation states and initial positions of active site water molecules, and compared with the existing crystallographic data of PTP1B. In WPD_closed conformation, the active site is found to maintain its conformation only in the protonated state of Asp181 in both free and liganded states, while Asp181 is likely to be deprotonated in WPD_open conformation. When the active site water molecule network that is a part of the free WPD_closed crystal structure is disrupted, intermediate WPD loop conformations, similar to that in the PTPRR crystal structure, are sampled in the MD simulations. In liganded PTP1B, one active site water molecule is found to be important for facilitating the orientation of Cys215 and the phosphate ion, thus may play a role in the reaction. In conclusion, conformational stability of WPD loop, and possibly catalytic activity of PTP1B, is significantly affected by the protonation state of Asp181 and position of active site water molecules, showing that these aspects should be taken into consideration both in MD simulations and inhibitor design. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Transition state analysis and requirement of Asp-262 general acid/base catalyst for full activation of dual-specificity phosphatase MKP3 by extracellular regulated kinase

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 Br?nsted 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.     

Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2

PHOSPHO1, a phosphoethanolamine/phosphocholine phosphatase, is upregulated in mineralising cells and is thought to be involved in the generation of inorganic phosphate for bone mineralisation. PHOSPHO2 is a putative phosphatase sharing 42% sequence identity with PHOSPHO1. Both proteins contain three catalytic motifs, conserved within the haloacid dehalogenase superfamily. Mutation of Asp32 and Asp203, key residues within two motifs, abolish PHOSPHO1 activity and confirm it as a member of this superfamily. We also show that Asp43 and Asp123, residues that line the substrate-binding site in our PHOSPHO1 model, are important for substrate hydrolysis. Further comparative modelling reveals that the active sites of PHOSPHO1 and PHOSPHO2 are very similar, but surprisingly, recombinant PHOSPHO2 hydrolyses phosphoethanolamine and phosphocholine relatively poorly. Instead, PHOSPHO2 shows high specific activity toward pyridoxal-5-phosphate (V(max) of 633 nmol min(-1) mg(-1) and K(m) of 45.5 microM). Models of PHOSPHO2 and PHOSPHO1 suggest subtle differences in the charge distributions around the putative substrate entry site and in the location of potential H-bond donors.     

Defining the active site of Schizosaccharomyces pombe C-terminal domain phosphatase Fcp1

Fcp1 is an essential protein serine phosphatase that dephosphorylates the C-terminal domain (CTD) of RNA polymerase II. By testing the effects of serial N- and C-terminal deletions of the 723-amino acid Schizosaccharomyces pombe Fcp1, we defined a minimal phosphatase domain spanning amino acids 156-580. We employed site-directed mutagenesis (introducing 24 mutations at 14 conserved positions) to locate candidate catalytic residues. We found that alanine substitutions for Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298) abrogated the phosphatase activity with either p-nitrophenyl phosphate or CTD-PO(4) as substrates. Structure-activity relationships were determined by introducing conservative substitutions at each essential position. Our results, together with previous mutational studies, highlight a constellation of seven amino acids (Asp(170), Asp(172), Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298)) that are conserved in all Fcp1 orthologs and likely comprise the active site. Five of these residues (Asp(170), Asp(172), Lys(280), Asp(297), and Asp(298)) are conserved at the active site of T4 polynucleotide 3'-phosphatase, suggesting that Fcp1 and T4 phosphatase are structurally and mechanistically related members of the DXD phosphotransferase superfamily.     

Solution structure of ERK2 binding domain of MAPK phosphatase MKP-3: structural insights into MKP-3 activation by ERK2

MAP kinases (MAPKs), which control mitogenic signal transduction in all eukaryotic organisms, are inactivated by dual specificity MAPK phosphatases (MKPs). MKP-3, a prototypical MKP, achieves substrate specificity through its N-terminal domain binding to the MAPK ERK2, resulting in the activation of its C-terminal phosphatase domain. The solution structure and biochemical analysis of the ERK2 binding (EB) domain of MKP-3 show that regions that are essential for ERK2 binding partly overlap with its sites that interact with the C-terminal catalytic domain, and that these interactions are functionally coupled to the active site residues of MKP-3. Our findings suggest a novel mechanism by which the EB domain binding to ERK2 is transduced to cause a conformational change of the C-terminal catalytic domain, resulting in the enzymatic activation of MKP-3.     

Prodomain processing of Asp1 (BACE2) is autocatalytic

Generation of the amyloid peptide through proteolytic processing of the amyloid precursor protein by beta- and gamma-secretases is central to the etiology of Alzheimer's disease. The highly elusive beta-secretase was recently identified as a transmembrane aspartic proteinase, Asp2 (BACE). The Asp2 homolog Asp1 (BACE2/DRAP) has also been reported to exhibit beta-secretase cleavage of amyloid precursor protein. Most aspartic proteinases are generated as inactive proenzymes, requiring removal of the prodomain to generate active proteinase. Here we show that prodomain processing of Asp1 occurs between Leu(62) and Ala(63) and is autocatalytic. Asp1 cleaved a maltose-binding protein-Asp1 prodomain fusion protein and a synthetic peptide at this site. Mutation of one of the conserved catalytic aspartic acid residues in the active site of Asp1 to asparagine (D110N) abolished this cleavage. Mutation of P(1)' and P(2)' residues in the substrate to phenylalanine reduced cleavage at this site. Asp1 expressed in cells was the mature form, and prodomain processing occurred intramolecularly within the endoplasmic reticulum/early Golgi. Interestingly, a proportion of mature Asp1 was expressed on the cell surface. When full-length Asp1(D110N) was expressed in COS-7 cells, it was not processed, suggesting that no other proteinase can activate Asp1 in these cells.     

3-D structure of a mutant (Asp101-->Ser) of E.coli alkaline phosphatase with higher catalytic activity.

Mutagenesis of the absolutely conserved residue Asp101 of the non-specific monoesterase alkaline phosphatase (E.C. 3.1.3.1) from E. coli has produced an enzyme with increased kcat. The carboxyl group of the Asp101 residue has been proposed to be involved in the positioning of Arg166 and the formation of the helix that contains the active site Ser102. The crystal structure of the Asp101-->Ser mutant has been refined at 2.5 A to a final crystallographic R-factor of 0.173. The altered active site structure of the mutant is compared with that of the wild-type as well as with the structures of the mutant enzyme soaked in two known alkaline phosphatase inhibitors (inorganic phosphate and arsenate). The changes affect primarily the side chain of Arg166 which, by losing the hydrogen bond interaction with the carboxyl side chain of Asp101, becomes more flexible. This analysis, in conjunction with product inhibition studies of the mutant enzyme, suggests that at high pH (> 7) the enzyme achieves a quicker catalytic turnover by allowing a faster release of the product.     

Design and characterization of an improved protein tyrosine phosphatase substrate-trapping mutant

Although members of the protein tyrosine phosphatase (PTPase) family share a common mechanism of action (hydrolysis of phosphotyrosine), the cellular processes in which they are involved can be both highly specialized and fundamentally important. Identification of cellular PTPase substrates will help elucidate the biological functions of individual PTPases. Two types of substrate-trapping mutants are being used to isolate PTPase substrates. In the first, the active site Cys residue is replaced by a Ser (e.g., PTP1B/C215S) while in the second, the general acid Asp residue is substituted by an Ala (e.g., PTP1B/D181A). Unfortunately, only a limited number of PTPase substrates have been identified with these two mutants, which are usually relatively abundant cellular proteins. Based on mechanistic considerations, we seek to create novel PTPase mutants with improved substrate-trapping properties. Kinetic and thermodynamic characterization of the newly designed PTP1B mutants indicates that PTP1B/D181A/Q262A displays lower catalytic activity than that of D181A. In addition, D181A/Q262A also possesses 6- and 28-fold higher substrate-binding affinity than those of D181A and C215S, respectively. In vivo substrate-trapping experiments indicate that D181A/Q262A exhibits much higher affinity than both D181A and C215S for a bona fide PTP1B substrate, the epidermal growth factor receptor. Moreover, D181A/Q262A can also identify novel, less abundant substrates, that are missed by D181A. Thus, this newly developed and improved substrate-trapping mutant can serve as a powerful affinity reagent to isolate and purify both high- and low-abundant protein substrates. Given that both Asp181 and Gln262 are invariant among the PTPase family, it is predicted that this improved substrate-trapping mutant would be applicable to all members of PTPases for substrate identification.     

Exchange of active site residues alters substrate specificity in extremely thermostable β-glycosidase from Thermococcus kodakarensis KOD1

β-Glycosidase from Thermococcus kodakarensis KOD1 is a hyperthermophilic enzyme with β-glucosidase, β-mannosidase, β-fucosidase and β-galactosidase activities. Sequence alignment with other β-glycosidases from hyperthermophilic archaea showed two unique active site residues, Gln77 and Asp206. These residues were represented by Arg and Asp in all other hyperthermophilic β-glycosidases. The two active site residues were mutated to Q77R, D206N and D206Q, to study the role of these unique active site residues in catalytic activity and to alter the substrate specificity to enhance its β-glucosidase activity. The secondary structure analysis of all the mutants showed no change in their structure and exhibited in similar conformation like wild-type as they all existed in dimer form in an SDS-PAGE under non-reducing conditions. Q77R and D206Q affected the catalytic activity of the enzyme whereas the D206N altered the catalytic turn-over rate for glucosidase and mannosidase activities with fucosidase activity remain unchanged. Gln77 is reported to interact with catalytic nucleophile and Asp206 with axial C2-hydroxyl group of substrates. Q77R might have made some changes in three dimensional structure due to its electrostatic effect and lost its catalytic activity. The extended side chains of D206Q is predicted to affect the substrate binding during catalysis. The high-catalytic turn-over rate by D206N for β-glucosidase activity makes it a useful enzyme in cellulose degradation at high temperatures.     

Mechanism of mitogen-activated protein kinase phosphatase-3 activation by ERK2

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.     

Catalytic cycling in beta-phosphoglucomutase: a kinetic and structural analysis

Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.     

A molecular dynamics study of the structural stability of HIV-1 protease under physiological conditions: the role of Na+ ions in stabilizing the active site

HIV-1 protease is most active under weakly acidic conditions (pH 3.5-6.5), when the catalytic Asp25 and Asp25' residues share 1 proton. At neutral pH, this proton is lost and the stability of the structure is reduced. Here we present an investigation of the effect of pH on the dynamics of HIV-1 protease using MD simulation techniques. MD simulations of the solvated HIV-1 protease with the Asp25/25' residues monoprotonated and deprotonated have been performed. In addition we investigated the effect of the inclusion of Na(+) and Cl(-) ions to mimic physiological salt conditions. The simulations of the monoprotonated form and deprotonated form including Na(+) show very similar behavior. In both cases the protein remained stable in the compact, "self-blocked" conformation in which the active site is blocked by the tips of the flaps. In the deprotonated system a Na(+) ion binds tightly to the catalytic dyad shielding the repulsion between the COO(-) groups. Ab initio calculations also suggest the geometry of the active site with the Na(+) bound closely resembles that of the monoprotonated case. In the simulations of the deprotonated form (without Na(+) ions), a water molecule bound between the Asp25 Asp25' side-chains. This disrupted the dimerization interface and eventually led to a fully open conformation.     

The active site base controls cofactor reactivity in Escherichia coli amine oxidase: x-ray crystallographic studies with mutational variants.

Amine oxidases utilize a proton abstraction mechanism following binding of the amine substrate to the C5 position of the cofactor, the quinone form of trihydroxyphenylalanine (TPQ). Previous work [Wilmot, C. M., et al. (1997) Biochemistry 36, 1608-1620] has shown that Asp383 in Escherichia coliamine oxidase (ECAO) is the catalytic base which performs the key step of proton abstraction. This paper explores in more depth this and other roles of Asp383. The crystal structures of three mutational variants are presented together with their catalytic properties, visible spectra, and binding properties for a substrate-like inhibitor, 2-hydrazinopyridine (2-HP), in comparison to those of the wild type enzyme. In wild type ECAO, the TPQ is located in a wedge-shaped pocket which allows more freedom of movement at the substrate binding position (C5) than for TPQ ring carbons C1-C4. A role of Asp383, whose carboxylate is located close to O5, is to stabilize the TPQ in its major conformation in the pocket. Replacement of Asp383 with the isostructural, but chemically distinct, Asn383 does not affect the location or dynamics of the TPQ cofactor significantly, but eliminates catalytic activity and drastically reduces the affinity for 2-HP. Removal of the side chain carboxyl moiety, as in Ala383, additionally allows the TPQ the greater conformational flexibility to coordinate to the copper, which demonstrates that Asp383 helps maintain the active site structure by preventing TPQ from migrating to the copper. Glu383 has a greatly decreased catalytic activity, as well as a decreased affinity for 2-HP relative to that of wild type ECAO. The electron density reveals that the longer side chain of Glu prevents the pivotal motion of the TPQ by hindering its movement within the wedge-shaped active site pocket. The results show that Asp383 performs multiple roles in the catalytic mechanism of ECAO, not only in acting as the active site base at different stages of the catalytic cycle but also in regulating the mobility of the TPQ that is essential to catalysis.