scholarly journals Characterization of recombinant fructose-1,6-bisphosphatase gene mutations: evidence of inhibition/activation of FBPase protein by gene mutation

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Gemma Topaz ◽  
Victor Epiter-Smith ◽  
Cristina Robalo ◽  
Megan Emad ◽  
Vanessa Ford ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Gene Mutations ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Coding Region ◽  
Functional Relationships ◽  
Fructose 1,6 Bisphosphatase ◽  
Natural Inhibitors

Abstract Specific residues of the highly regulated fructose-1,6-bisphosphatase (FBPase) enzyme serve as important contributors to the catalytic activity of the enzyme. Previous clinical studies exploring the genetic basis of hypoglycemia revealed two significant mutations in the coding region of the FBPase gene in patients with hypoglycemia, linking the AMP-binding site to the active site of the enzyme. In the present study, a full kinetic analysis of similar mutants was performed. Kinetic results of mutants Y164A and M177A revealed an approximate two to three-fold decrease in inhibitory constants (Ki’s) for natural inhibitors AMP and fructose-2,6-bisphosphate (F2,6-BP) compared with the Wild-type enzyme (WT). A separate mutation (M248D) was performed in the active site of the enzyme to investigate whether the enzyme could be activated. This mutant displayed an approximate seven-fold increase in Ki for F2,6-BP. Interfacial mutants L56A and L73A exhibited an increase in Ki for F2,6-BP by approximately five-fold. Mutations in the AMP-binding site (K112A and Y113A) demonstrated an eight to nine-fold decrease in AMP inhibition. Additionally, mutant M248D displayed a four-fold decrease in its apparent Michelis constant (Km), and a six-fold increase in catalytic efficiency (CE). The importance—and medical relevance—of specific residues for FBPase structural/functional relationships in both the catalytic site and AMP-binding site is discussed.

scholarly journals Structural and biochemical characterization of the class II fructose-1,6-bisphosphatase from Francisella tularensis

2020 ◽  
Vol 76 (11) ◽  
pp. 524-535
Author(s):  
Anna I. Selezneva ◽  
Hiten J. Gutka ◽  
Nina M. Wolf ◽  
Fnu Qurratulain ◽  
Farahnaz Movahedzadeh ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Active Site ◽  
Biochemical Characterization ◽  
Structural Characteristics ◽  
Crystal Form ◽  
Class Ii ◽  
Monoclinic Crystal ◽  
Cell Parameters ◽  
Functional Relationships ◽  
Fructose 1,6 Bisphosphatase

The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Å resolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02 Å, β = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D 2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower K m and V max than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.

scholarly journals Fructose 1,6-bisphosphatase: getting the message across

2019 ◽  
Vol 39 (3) ◽  
Author(s):  
David J. Timson
Keyword(s):  
Information Transmission ◽  
Binding Site ◽  
Metal Ion ◽  
Drug Target ◽  
Fold Increase ◽  
Fructose 1,6 Bisphosphatase ◽  
Additional Detail ◽  
Key Enzyme ◽  
Mammalian Enzyme ◽  
The Warburg Effect

Abstract Fructose 1,6-bisphosphatase (FBPase) is a key enzyme in gluconeogenesis. It is a potential drug target in the treatment of type II diabetes. The protein is also associated with a rare inherited metabolic disease and some cancer cells lack FBPase activity which promotes glycolysis facilitating the Warburg effect. Thus, there is interest in both inhibiting the enzyme (for diabetes treatment) and restoring its activity (in relevant cancers). The mammalian enzyme is tetrameric, competitively inhibited by Fructose 2,6-bisphosphate and negatively allosterically regulated by AMP. This allosteric regulation requires information transmission between the AMP binding site and the active site of the enzyme. A recent paper by Topaz et al. (Bioscience Reports (2019) 39, pii:BSR20180960) has added additional detail to our understanding of this information transmission process. Two residues in the AMP binding site (Lys112 and Tyr113) were shown to be involved in initiating the message between the two sites. This tyrosine residue has recently be shown to be important with protein’s interaction with the antidiabetic drug metformin. A variant designed to increase metal ion affinity (M248D) resulted in a five-fold increase in enzymatic activity. Interestingly alterations of two residues at the subunit interfaces (Tyr164 and Met177) resulted in increased responsiveness to AMP. Overall, these findings may have implications in the design of novel FBPase inhibitors or activators.

scholarly journals Purification and characterization of Ak.1 protease, a thermostable subtilisin with a disulphide bond in the substrate-binding cleft

2000 ◽  
Vol 350 (1) ◽  
pp. 321-328 ◽  
Author(s):  
Helen S. TOOGOOD ◽  
Clyde A. SMITH ◽  
Edward N. BAKER ◽  
Roy M. DANIEL
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Fold Increase ◽  
Branched Chain Amino Acids ◽  
Disulphide Bond ◽  
Purification And Characterization ◽  
Active Site Cleft ◽  
Binding Cleft ◽  
Branched Chain

Ak.1 protease, a thermostable subtilisin isolated originally from Bacillus st. Ak.1, was purified to homogeneity from the Escherichia coli clone PB5517. It is active against substrates containing neutral or hydrophobic branched-chain amino acids at the P1 site, such as valine, alanine or phenylalanine. The Km and kcat of the enzyme decrease with decreasing temperature, though not to the same degree with all substrates, suggesting that specificity changes with temperature. The protease is markedly stabilized by Ca2+ ions. At 70°C, a 10-fold increase in Ca2+ concentration increases the half-life by three orders of magnitude. Ak.1 protease is stabilized by Ca2+ to a greater extent than is thermitase. This may be due, in part, to the presence of an extra Ca2+-binding site in Ak.1 protease. Other metal ions, such as Sr2+, increase the thermostability of the enzyme, but to a significantly lower degree than does Ca2+. The structure of the protease showed the presence of a disulphide bond located within the active-site cleft. This bond influences both enzyme activity and thermostability. The disulphide bond appears to have a dual role: maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease. The protease was originally investigated to determine its usefulness in the clean-up of DNA at high temperatures. However, it was found that this protease has a limited substrate specificity, so this application was not explored further.

Structure and kinetic characterization of human sperm-specific glyceraldehyde-3-phosphate dehydrogenase, GAPDS

2011 ◽  
Vol 435 (2) ◽  
pp. 401-409 ◽  
Author(s):  
Apirat Chaikuad ◽  
Naeem Shafqat ◽  
Ruby Al-Mokhtar ◽  
Gus Cameron ◽  
Anthony R. Clarke ◽  
...  
Keyword(s):  
Hormonal Contraception ◽  
Anion Recognition ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Human Sperm ◽  
Glycolytic Enzyme ◽  
Soluble Form ◽  
Active Centre ◽  
Recognition Sites

hGAPDS (human sperm-specific glyceraldehyde-3-phosphate dehydrogenase) is a glycolytic enzyme essential for the survival of spermatozoa, and constitutes a potential target for non-hormonal contraception. However, enzyme characterization of GAPDS has been hampered by the difficulty in producing soluble recombinant protein. In the present study, we have overexpressed in Escherichia coli a highly soluble form of hGAPDS truncated at the N-terminus (hGAPDSΔN), and crystallized the homotetrameric enzyme in two ligand complexes. The hGAPDSΔN–NAD+–phosphate structure maps the two anion-recognition sites within the catalytic pocket that correspond to the conserved Ps site and the newly recognized Pi site identified in other organisms. The hGAPDSΔN–NAD+–glycerol structure shows serendipitous binding of glycerol at the Ps and new Pi sites, demonstrating the propensity of these anion-recognition sites to bind non-physiologically relevant ligands. A comparison of kinetic profiles between hGAPDSΔN and its somatic equivalent reveals a 3-fold increase in catalytic efficiency for hGAPDSΔN. This may be attributable to subtle amino acid substitutions peripheral to the active centre that influence the charge properties and protonation states of catalytic residues. Our data therefore elucidate structural and kinetic features of hGAPDS that might provide insightful information towards inhibitor development.

scholarly journals Anti-CUB1 or Anti-Spacer Antibodies That Increase ADAMTS13 Activity Act By Allosterically Enhancing Metalloprotease Domain Function

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 23-23
Author(s):  
An-Sofie Schelpe ◽  
Anastasis Petri ◽  
Nele Vandeputte ◽  
Hans Deckmyn ◽  
Simon F De Meyer ◽  
...  
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Active Site ◽  
Current Model ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Activation Mechanism ◽  
Rich Domain ◽  
Cryptic Epitope

Abstract Background ADAMTS13 circulates in a folded conformation, which is mediated by interactions between the C-terminal CUB domains and its central Spacer domain. Binding of ADAMTS13 to the VWF D4-CK domains disrupts the CUB-Spacer interaction, inducing a structural change that extends ADAMTS13 into an open conformation that enhances catalytic efficiency ~2-fold. This mechanism supports a model in which ADAMTS13 unfolding induces exposure of an exosite in the Spacer domain that interacts with the VWF A2 domain, increasing the affinity between the two molecules, and, therefore, the rate of proteolysis. The D4-CK-mediated conformational activation of ADAMTS13 can be mimicked in vitro with the use of antibodies that disrupt the CUB-Spacer interaction, such as the previously published anti-CUB antibody, Ab17G2. We recently generated a novel, activating antibody against the Spacer domain (Ab3E4). Aim To characterize the mechanism by which the Ab17G2 and Ab3E4 enhance the catalytic efficiency of ADAMTS13. Methods The effects of the Ab17G2 and Ab3E4 on the activity of ADAMTS13 were studied using FRETS-VWF73. The effects of the Ab17G2 and Ab3E4 on the kinetics of VWF96 (VWF G1573-R1668) proteolysis were characterized using an in-house assay. ELISA was used to investigate conformational changes in ADAMTS13 induced by the Ab17G2 and Ab3E4. Results Both Ab17G2 and Ab3E4 enhanced FRETS-VWF73 proteolysis by ~1.7-fold. This result was reproduced using the VWF96 substrate; the Ab17G2 and Ab3E4 enhanced the catalytic efficiency (kcat/Km) of ADAMTS13 by ~1.8- and ~2.0-fold, respectively. The activation was dependent on the conformational extension of ADAMTS13, since the antibodies could not enhance the activity of an ADAMTS13 variant that lacks the TSP2-CUB2 domains (MDTCS). Surprisingly, ADAMTS13 activation was not mediated through exposure of the Spacer or Cys-rich domain exosites as previously proposed, as the Ab17G2 and Ab3E4 efficiently enhanced proteolysis of VWF96 variants in which the Spacer/Cys-rich exosite binding sites were disrupted. Kinetic analysis of VWF96 proteolysis showed that the Ab17G2- and Ab3E4-induced activation of ADAMTS13 is primarily manifest through a ~1.5- to ~2-fold increase in enzyme turnover (kcat). Thus, contrary to the current model, this suggests that the conformational extension of ADAMTS13 influences the functionality of the active site, and not substrate binding affinity (Km). Incubating ADAMTS13 with either Ab17G2 or Ab3E4 exposed a cryptic epitope in the metalloprotease domain that was specifically detected by ELISA, further corroborating that the antibodies induce a conformational change in ADAMTS13 affecting the M domain. Conclusion Antibodies can be used as tools for understanding the structure/function of enzymes. Using activating antibodies against the Spacer and CUB1 domains of ADAMTS13, we show for the first time that the activation of ADAMTS13 following its unfolding is not a result of exposure of a functional exosite in Spacer/Cys-rich domain that increases affinity to VWF. Rather, our data are consistent with an allosteric activation mechanism upon the metalloprotease domain. We propose that ADAMTS13 unfolding causes a conformational change in the active site that further activates the enzyme. We are currently investigating whether the D4-CK-induced enhancement of ADAMTS13 proteolytic activity is also mediated by conformational changes in the active site. Disclosures Vanhoorelbeke: Ablynx: Consultancy; Shire: Consultancy.

scholarly journals Reassessment of the Transhydrogenase/Malate Shunt Pathway in Clostridium thermocellum ATCC 27405 through Kinetic Characterization of Malic Enzyme and Malate Dehydrogenase

2015 ◽  
Vol 81 (7) ◽  
pp. 2423-2432 ◽  
Author(s):  
M. Taillefer ◽  
T. Rydzak ◽  
D. B. Levin ◽  
I. J. Oresnik ◽  
R. Sparling
Keyword(s):  
Malate Dehydrogenase ◽  
Malic Enzyme ◽  
Clostridium Thermocellum ◽  
Consolidated Bioprocessing ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Cellulosic Biomass ◽  
Content Type ◽  
Malic Enzyme Activity

ABSTRACTClostridium thermocellumproduces ethanol as one of its major end products from direct fermentation of cellulosic biomass. Therefore, it is viewed as an attractive model for the production of biofuels via consolidated bioprocessing. However, a better understanding of the metabolic pathways, along with their putative regulation, could lead to improved strategies for increasing the production of ethanol. In the absence of an annotated pyruvate kinase in the genome, alternate means of generating pyruvate have been sought. Previous proteomic and transcriptomic work detected high levels of a malate dehydrogenase and malic enzyme, which may be used as part of a malate shunt for the generation of pyruvate from phosphoenolpyruvate. The purification and characterization of the malate dehydrogenase and malic enzyme are described in order to elucidate their putative roles in malate shunt and their potential role inC. thermocellummetabolism. The malate dehydrogenase catalyzed the reduction of oxaloacetate to malate utilizing NADH or NADPH with akcatof 45.8 s−1or 14.9 s−1, respectively, resulting in a 12-fold increase in catalytic efficiency when using NADH over NADPH. The malic enzyme displayed reversible malate decarboxylation activity with akcatof 520.8 s−1. The malic enzyme used NADP+as a cofactor along with NH4+and Mn2+as activators. Pyrophosphate was found to be a potent inhibitor of malic enzyme activity, with aKiof 0.036 mM. We propose a putative regulatory mechanism of the malate shunt by pyrophosphate and NH4+based on the characterization of the malate dehydrogenase and malic enzyme.

scholarly journals Characterization of the membrane quinoprotein glucose dehydrogenase from Escherichia coli and characterization of a site-directed mutant in which histidine-262 has been changed to tyrosine

1999 ◽  
Vol 340 (3) ◽  
pp. 639-647 ◽  
Author(s):  
Gyles E. COZIER ◽  
Raff A. SALLEH ◽  
Christopher ANTHONY
Keyword(s):  
Escherichia Coli ◽  
Electron Transfer ◽  
Active Site ◽  
Marked Decrease ◽  
Glucose Dehydrogenase ◽  
Catalytic Efficiency ◽  
Prosthetic Group ◽  
Competitive Inhibitors ◽  
A Site

The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH 7 and pH 8 being involved. Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process.

scholarly journals Generation and Characterization of a Mutant of Influenza A Virus Selected with the Neuraminidase Inhibitor BCX-140

1998 ◽  
Vol 42 (4) ◽  
pp. 801-807 ◽  
Author(s):  
Shanta Bantia ◽  
Anita A. Ghate ◽  
Sandya L. Ananth ◽  
Y. Sudhakar Babu ◽  
Gillian M. Air ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Binding Site ◽  
Active Site ◽  
Influenza A ◽  
Neuraminidase Inhibitor ◽  
Active Site Residues ◽  
Binding Studies ◽  
Drug Induced ◽  
Guanidino Group

ABSTRACT Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.

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