Phosphofructokinase: structure and control

1981 ◽  
Vol 293 (1063) ◽  
pp. 53-62 ◽  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Bacillus Stearothermophilus ◽  
Phosphoryl Transfer ◽  
Active Conformation ◽  
Allosteric Activation ◽  
Ligand Binding Sites ◽  
And Control ◽  
Allosteric Activator ◽  
Cooperative Kinetics

Phosphofructokinase from Bacillus stearothermophilus shows cooperative kinetics with respect to the substrate fructose-6-phosphate (F6P), allosteric activation by ADP, and inhibition by phosphoenolpyruvate. The crystal structure of the active conformation of the enzyme has been solved to 2.4 A resolution, and three ligand-binding sites have been located. Two of these form the active site and bind the substrates F6P and ATP. The third site binds both allosteric activator and inhibitor. The complex of the enzyme with F6P and ADP has been partly refined at 2.4 A resolution, and a model of ATP has been built into the active site by using the refined model of ADP and a 6 A resolution map of bound 5'-adenylylimidodiphosphate (AMPPNP). The y-phosphate of ATP is close to the 1-hydroxyl of F6P, in a suitable position for in-line phosphoryl transfer. The binding of the phosphate of F6P involves two arginines from a neighbouring subunit in the tetramer, which suggests that a rearrangement of the subunits could explain the cooperativity of substrate binding. The activator ADP is also bound by residues from two subunits.

NTP-driven translocation and regulation of downstream template opening by multi-subunit RNA polymerases

2005 ◽  
Vol 83 (4) ◽  
pp. 486-496 ◽  
Author(s):  
Zachary F Burton ◽  
Michael Feig ◽  
Xue Q Gong ◽  
Chunfen Zhang ◽  
Yuri A Nedialkov ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Site ◽  
Binding Sites ◽  
Rna Synthesis ◽  
Rna Polymerases ◽  
Phosphoryl Transfer ◽  
Elongation Complex ◽  
Detailed Model ◽  
Loop 2

Multi-subunit RNA polymerases bind nucleotide triphosphate (NTP) substrates in the pretranslocated state and carry the dNMP–NTP base pair into the active site for phosphoryl transfer. NTP-driven translocation requires that NTP substrates enter the main-enzyme channel before loading into the active site. Based on this model, a new view of fidelity and efficiency of RNA synthesis is proposed. The model predicts that, during processive elongation, NTP-driven translocation is coupled to a protein conformational change that allows pyrophosphate release: coupling the end of one bond-addition cycle to substrate loading and translocation for the next. We present a detailed model of the RNA polymerase II elongation complex based on 2 low-affinity NTP binding sites located in the main-enzyme channel. This model posits that NTP substrates, elongation factors, and the conserved Rpb2 subunit fork loop 2 cooperate to regulate opening of the downstream transcription bubble.Key words: RNA polymerase, NTP-driven translocation, transcriptional fidelity, transcriptional efficiency, α-amanitin.

scholarly journals Self-association configures the NAD+-binding site of plant NLR TIR domains

2021 ◽  
Author(s):  
Hayden Burdett ◽  
Xiahao Hu ◽  
Maxwell X Rank ◽  
Natsumi Maruta ◽  
Bostjan Kobe
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Active Site ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Structural Features ◽  
Active Conformation ◽  
Effector Triggered Immunity ◽  
Self Association ◽  
Tir Domains

TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD+ binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the ″AE interface″ (mediated by helices A and E), the ″BB-loop″ (connecting β-strand B and helix B in the structure), and the ″BE interface″ (mediated by the BB-loop from one TIR and the ″DE surface″ of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD+-binding site, shifting the BB-loop away from the catalytic site and allowing NAD+ to access the active site. We propose that an intact ″DE surface″ is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD+ or NADP+ is not sufficient to induce self-association, suggesting that NAD+ binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.

Molecular Modeling and Virtual Screening of Molecular Inhibitors for Leptospiral Collagenase

2021 ◽  
Author(s):  
Vikram Kumar ◽  
Nagesh Srikaku ◽  
Veeranarayanan Surya Aathmanathan ◽  
Padikara K Satheeshkumar ◽  
Madanan Gopalakrishnan Madathiparambil ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Molecular Modeling ◽  
Active Site ◽  
Binding Sites ◽  
Catalytic Site ◽  
Simulation Studies ◽  
C Terminus ◽  
Ligand Binding Sites ◽  
N Terminus ◽  
The Stability

Abstract Collagenase is a virulence factor which facilitates the invasion of pathogenic Leptospira into the host. In the present study, the model of Leptopsiral collagenase was constructed by employing threading method with the crystal structure of collagenase G. Three ligand binding sites at N- terminus, catalytic site and C-terminus were predicted by Metapocket server. Among sixty seven inhibitors from the ChEBI and Zinc databases, Protohypericin is predicted as the best inhibitor since it binds at the catalytic site of Leptopsiral collagenase. Molecular dynamic simulation studies validated the stability of interaction between the active site of Leptospiral collagenase and Protohypericin. The docking and molecular simulation studies corroborated the potential of the ligand to curb leptospiral infection.

Ligand Binding Sites inEscherichia coliInorganic Pyrophosphatase:  Effects of Active Site Mutations†

Biochemistry ◽  
2001 ◽  
Vol 40 (15) ◽  
pp. 4645-4653 ◽  
Author(s):  
Teppo Hyytiä ◽  
Pasi Halonen ◽  
Anu Salminen ◽  
Adrian Goldman ◽  
Reijo Lahti ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Active Site ◽  
Binding Sites ◽  
Ligand Binding Sites ◽  
Active Site Mutations

scholarly journals Second distinct conformation of the phosphohistidine loop in succinyl-CoA synthetase

2021 ◽  
Vol 77 (3) ◽  
pp. 357-368
Author(s):  
Ji Huang ◽  
Marie E. Fraser
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Citric Acid Cycle ◽  
Phosphoryl Group ◽  
Phosphoryl Transfer ◽  
Specific Enzyme ◽  
Substrate Level ◽  
Mechanism Of Catalysis ◽  
Active Site Histidine ◽  
Succinyl Coa Synthetase

Succinyl-CoA synthetase (SCS) catalyzes a reversible reaction that is the only substrate-level phosphorylation in the citric acid cycle. One of the essential steps for the transfer of the phosphoryl group involves the movement of the phosphohistidine loop between active site I, where CoA, succinate and phosphate bind, and active site II, where the nucleotide binds. Here, the first crystal structure of SCS revealing the conformation of the phosphohistidine loop in site II of the porcine GTP-specific enzyme is presented. The phosphoryl transfer bridges a distance of 29 Å between the binding sites for phosphohistidine in site I and site II, so these crystal structures support the proposed mechanism of catalysis by SCS. In addition, a second succinate-binding site was discovered at the interface between the α- and β-subunits of SCS, and another magnesium ion was found that interacts with the side chains of Glu141β and Glu204β via water-mediated interactions. These glutamate residues interact with the active-site histidine residue when it is bound in site II.

scholarly journals Allosteric activation of brain hexokinase by magnesium ions and by magnesium-ion–adenosine triphosphate complex

1971 ◽  
Vol 125 (1) ◽  
pp. 249-254 ◽  
Author(s):  
H. S. Bachelard
Keyword(s):  
Binding Sites ◽  
Magnesium Ion ◽  
Magnesium Ions ◽  
Allosteric Activation ◽  
Substrate Saturation ◽  
Number Of Binding Sites ◽  
High Concentrations ◽  
Substrate Binding Sites ◽  
Hill Plots ◽  
Allosteric Activator

1. Substrate-saturation curves of brain hexokinase for MgATP2− were sigmoidal at sub-saturating concentrations of glucose when the Mg2+/ATP ratio was maintained at 1:1. Under identical conditions, except that Mg2+ was present in excess, hyperbolic curves were observed. 2. The number of binding sites (calculated from Hill plots) is 1.8 at a Mg2+/ATP ratio 1:1, and 1.0 with excess of Mg2+. The apparent Km for MgATP2− is 6.5×10−4m at a Mg2+/ATP ratio 1:1, and 3.5×10−4m with excess of Mg2+. 3. Interdependence between substrate-binding sites was indicated by the effects of varying the concentration of glucose. The sigmoidality and deviation from Michaelis–Menten kinetics at a Mg2+/ATP ratio 1:1 became less pronounced with increasing glucose concentration. Also, although substrate-saturation curves for glucose were hyperbolic when the Mg2+/ATP ratio was 1:1, reciprocal plots were non-linear. These were linear with excess of Mg2+. 4. High concentrations of Mg2+ (Mg2+/ATP ratios above 5:1) were inhibitory. 5. The results are taken to indicate homotropic co-operative binding of MgATP2− and that Mg2+ is an allosteric activator. Possible implications in regulation are discussed.

scholarly journals Crystallographic fragment screening-based study of a novel FAD-dependent oxidoreductase from Chaetomium thermophilum

2021 ◽  
Vol 77 (6) ◽  
Author(s):  
Leona Švecová ◽  
Lars Henrik Østergaard ◽  
Tereza Skálová ◽  
Kirk Matthew Schnorr ◽  
Tomáš Koval' ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Enzyme Catalysis ◽  
High Throughput Screening ◽  
Fragment Screening ◽  
Active Site Pocket ◽  
Chaetomium Thermophilum ◽  
The Family ◽  
Ligand Binding Sites ◽  
Gmc Oxidoreductase

The FAD-dependent oxidoreductase from Chaetomium thermophilum (CtFDO) is a novel thermostable glycoprotein from the glucose–methanol–choline (GMC) oxidoreductase superfamily. However, CtFDO shows no activity toward the typical substrates of the family and high-throughput screening with around 1000 compounds did not yield any strongly reacting substrate. Therefore, protein crystallography, including crystallographic fragment screening, with 42 fragments and 37 other compounds was used to describe the ligand-binding sites of CtFDO and to characterize the nature of its substrate. The structure of CtFDO reveals an unusually wide-open solvent-accessible active-site pocket with a unique His–Ser amino-acid pair putatively involved in enzyme catalysis. A series of six crystal structures of CtFDO complexes revealed five different subsites for the binding of aryl moieties inside the active-site pocket and conformational flexibility of the interacting amino acids when adapting to a particular ligand. The protein is capable of binding complex polyaromatic substrates of molecular weight greater than 500 Da.

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