scholarly journals The roles of the conserved pyrimidine bases in hammerhead ribozyme catalysis: evidence for a magnesium ion-binding site

1995 ◽  
Vol 311 (2) ◽  
pp. 487-494 ◽  
Author(s):  
J B Murray ◽  
C J Adams ◽  
J R P Arnold ◽  
P G Stockley
Keyword(s):  
Binding Site ◽  
Hammerhead Ribozyme ◽  
Fold Increase ◽  
Magnesium Ion ◽  
Ion Binding ◽  
Kinetic Properties ◽  
Direct Role ◽  
Single Turnover ◽  
Catalytically Active ◽  
Pyrimidine Bases

We report details of the synthesis and characterization of oligoribonucleotides containing 4-thiouridine or 2-pyrimidinone ribonucleoside (4HC). We have used these probes to examine the roles of the conserved pyrimidines in the central core of the hammerhead ribozyme. The effects on catalysis of singly-substituted hammerhead ribozyme and substrate strands were quantified in multiple-turnover reactions. Various effects were observed on kcat. and Km, with up to a 7-fold decrease and a 3-fold increase respectively. For substitutions with 4HC at positions 3 or 17, catalytic activity in single turnover reactions can be increased up to 8-fold equivalent to 40% of wild-type activity, by increasing the concentration of the Mg2+ cofactor, implying that these substitutions had a deleterious effect on Mg2+ binding. Calculations of the change in the apparent free energy of binding for variants at positions 3, 4 or 17 are each consistent with deletion of a single hydrogen-bond to an uncharged group in the ribozyme. The cytidine 5′ to the scissile phosphate had not previously been thought to play a direct role in catalysis, however, removal of the exocyclic amino group decreased kcat. 4-fold. Recently, the crystal structures of a hammerhead ribozyme bound to either a non-cleavable 2′-deoxy substrate strand or a ribo-substrate strand have been reported. The kinetic properties of the variants described here are consistent with several key interactions seen in the crystals, in particular they provide experimental support for the assignment of the proposed catalytically active magnesium ion-binding site.

scholarly journals Insolubilized enzymes. Kinetic behaviour of glucose oxidase bound to porous glass particles

1971 ◽  
Vol 124 (4) ◽  
pp. 801-807 ◽  
Author(s):  
Michael K. Weibel ◽  
Harold J. Bright
Keyword(s):  
Glucose Oxidase ◽  
Porous Glass ◽  
Fold Increase ◽  
Kinetic Mechanism ◽  
Diffusion Control ◽  
Kinetic Properties ◽  
Substrate Diffusion ◽  
Catalytically Active ◽  
Absolute Magnitudes ◽  
Covalently Linked

1. The spectrophotometric and steady-state kinetic properties of glucose oxidase (EC 1.1.3.4, from Aspergillus niger) that is covalently linked to porous glass beads have been examined. These properties have been compared with those of soluble glucose oxidase, for which the kinetic mechanism at pH5.5 and 25°C has been established previously by a combination of conventional and rapid-reaction techniques to be the following: [Formula: see text] where Eo and Er represent oxidized and reduced forms of the enzyme, respectively. 2. The ratio k+4/k+2 is unchanged after insolubilization, and evidence is presented which suggests that the absolute magnitudes of k+4 and k+2 are unchanged. 3. The kinetic efficiency of the insolubilized enzyme is greatly enhanced because of a 14-fold increase in the apparent affinity of glucose for Eo. This effect is attributed either to the binding of glucose to the glass surface or to a change in enzyme structure imposed by the insolubilization process. 4. Only 6% of the insolubilized enzyme which can be reduced by glucose is catalytically active. It is shown by calculation and direct experimental evidence that this fraction of catalytically active enzyme is bound to the exterior bead surface. The remaining 94% of the enzyme is bound within the pore network and may be subject to severe substrate diffusion control.

Identification and Characterization of a Sodium Ion Binding Site on the Staphylocoagulase-Prothrombin Complex.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1700-1700
Author(s):  
Peter R. Panizzi ◽  
Paul E. Bock
Keyword(s):  
Binding Site ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Serine Proteinase ◽  
Fold Increase ◽  
Sodium Ion ◽  
Ion Binding ◽  
Chromogenic Substrate ◽  
T Complex ◽  
Enhanced Activity

Abstract Sodium ion binding regulates allosterically the pro- and anti-coagulant functions of the central coagulation serine proteinase, thrombin (T). The conversion of the prothrombin (ProT) zymogen into T results in expression of the fibrinogen (Fbg) recognition site (exosite I) and may facilitate binding of a single Na+. Na+ binding stabilizes the “fast” form of T that mediates the efficient conversion of Fbg into fibrin. The “slow” form of T, lacking Na+, shows decreased Fbg substrate activity and slightly enhanced activity towards activation of protein C, which results in a net increase in the anticoagulant activity of T (Di Cera et al., 1995). Conversion of protein C to activated protein C results in cleavage and inactivation of factor Va and VIIIa, which inhibits reactions central to hemostasis. Staphylocoagulase (SC), a conformational activator of ProT, binds ProT with high affinity through proexosite I and imparts T-like activity to the zymogen-activator complex. To determine whether the Na+ binding site is present on the SC-(1-325)·ProT complex, kinetic studies in the presence and absence of Na+ were performed, using the non-interacting cation, choline to maintain constant ionic strength. Results indicate that indeed Na+ binds to the SC-(1-325)·ProT complex with a KD of 17 ± 2 mM and causes an ~7-fold increase in the activity of SC-(1-325)·ProT complex toward a T-specific tripeptide chromogenic substrate. The chromogenic substrate activity of mouse ProT·SC-(1-325), unlike the human zymogen, was independent of Na+, consistent with the recent finding that mouse T lacks a functional Na+ binding site (Bush et al., 2006). Fbg clotting studies indicated that human T has 24-fold increased clotting activity in the presence of Na+, and the SC-(1-325)·T complex has 13-fold enhanced activity. Surprisingly, much unlike the results obtained from the chromogenic substrate studies, the Fbg clotting activity of the SC-(1-325)·ProThuman complex was less dependent on the presence of Na+, as indicated by a 4.1-fold increase in clotting activity for the complex in the presence of Na+. Further studies will be necessary to elucidate the functional significance of the Na+ binding site on the SC·ProT complex and its effect on Fbg recognition and cleavage.

Identification of the Magnesium Ion Binding Site in the Catalytic Center ofEscherichiacoliPrimase by Iron Cleavage†

Biochemistry ◽  
2000 ◽  
Vol 39 (2) ◽  
pp. 332-339 ◽  
Author(s):  
G. Nigel Godson ◽  
Jurek Schoenich ◽  
Wuliang Sun ◽  
A. Arkady Mustaev
Keyword(s):  
Binding Site ◽  
Magnesium Ion ◽  
Ion Binding ◽  
Catalytic Center

Identification of the Hammerhead Ribozyme Metal Ion Binding Site Responsible for Rescue of the Deleterious Effect of a Cleavage Site Phosphorothioate†

Biochemistry ◽  
1999 ◽  
Vol 38 (43) ◽  
pp. 14363-14378 ◽  
Author(s):  
Shenglong Wang ◽  
Katrin Karbstein ◽  
Alessio Peracchi ◽  
Leonid Beigelman ◽  
Daniel Herschlag
Keyword(s):  
Binding Site ◽  
Cleavage Site ◽  
Metal Ion ◽  
Deleterious Effect ◽  
Hammerhead Ribozyme ◽  
Ion Binding ◽  
Metal Ion Binding

Functional Characterization of Ion Permeation Pathway in the N-Type Ca2+ Channel

1998 ◽  
Vol 79 (2) ◽  
pp. 622-634 ◽  
Author(s):  
Minoru Wakamori ◽  
Mark Strobeck ◽  
Tetsuhiro Niidome ◽  
Tetsuyuki Teramoto ◽  
Keiji Imoto ◽  
...  
Keyword(s):  
Binding Site ◽  
High Voltage ◽  
Functional Characterization ◽  
Ion Binding ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Ion Permeation ◽  
Patch Clamp Technique ◽  
Homogeneous Population

Wakamori, Minoru, Mark Strobeck, Tetsuhiro Niidome, Tetsuyuki Teramoto, Keiji Imoto, and Yasuo Mori. Functional characterization of ion permeation pathway in the N-type Ca2+ channel. J. Neurophysiol. 79: 622–634, 1998. Multiple types of high-voltage-activated Ca2+ channels, including L-, N-, P-, Q- and R-types have been distinguished from each other mainly employing pharmacological agents that selectively block particular types of Ca2+ channels. Except for the dihydropyridine-sensitive L-type Ca2+ channels, electrophysiological characterization has yet to be conducted thoroughly enough to biophysically distinguish the remaining Ca2+ channel types. In particular, the ion permeation properties of N-type Ca2+ channels have not been clarified, although the kinetic properties of both the L- and N-type Ca2+ channels are relatively well described. To establish ion conducting properties of the N-type Ca2+ channel, we examined a homogeneous population of recombinant N-type Ca2+ channels expressed in baby hamster kidney cells, using a conventional whole cell patch-clamp technique. The recombinant N-type Ca2+ channel, composed of the α1B, α2a, and β1a subunits, displayed high-voltage-activated Ba2+ currents elicited by a test pulse more positive than −30 mV, and were strongly blocked by the N-type channel blocker ω-conotoxin-GVIA. In the presence of 110 mM Ba2+, the unitary current showed a slope conductance of 18.2 pS, characteristic of N-type channels. Ca2+ and Sr2+ resulted in smaller ion fluxes than Ba2+, with the ratio 1.0:0.72:0.75 of maximum conductance in current-voltage relationships of Ba2+, Ca2+, and Sr2+ currents, respectively. In mixtures of Ba2+ and Ca2+, where the Ca2+ concentration was steadily increased in place of Ba2+, with the total concentration of Ba2+ and Ca2+ held constant at 3 mM, the current amplitude went through a clear minimum when 20% of the external Ba2+ was replaced by Ca+2. This anomalous mole fraction effect suggests an ion-binding site where two or more permeant ions can sit simultaneously. By using an external solution containing 110 mM Na+ without polyvalent cations, inward Na+ currents were evoked by test potentials more positive than −50 mV. These currents were activated and inactivated in a kinetic manner similar to that of Ba2+ currents. Application of inorganic Ca2+ antagonists blocked Ba2+ currents through N-type channels in a concentration-dependent manner. The rank order of inhibition was La3+ ≥ Cd2+ ≫ Zn2+ > Ni2+ ≥ Co2+. When a short strong depolarization was applied before test pulses of moderate depolarizing potentials, relief from channel blockade by La3+ and Cd2+ and subsequent channel reblocking was observed. The measured rate (2 × 108 M−1 s−1) of reblocking approached the diffusion-controlled limit. These results suggest that N-type Ca2+ channels share general features of a high affinity ion-binding site with the L-type Ca2+ channel, and that this site is easily accessible from the outside of the channel pore.

scholarly journals Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

2006 ◽  
Vol 400 (3) ◽  
pp. 385-392 ◽  
Author(s):  
Erdeni Bai ◽  
Federico I. Rosell ◽  
Bao Lige ◽  
Marcia R. Mauk ◽  
Barbara Lelj-Garolla ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Metal Ions ◽  
Binding Site ◽  
Metal Ion ◽  
Association Constants ◽  
Ion Binding ◽  
Ferric Uptake Regulator ◽  
Metal Ion Binding ◽  
Dimerization Domain ◽  
High Affinity

The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (KA) of 10(±7)×106, 5.7(±3)×106, 2.0(±2)×106 and 2.0(±3)×104 M−1 for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(±2)×106, 3.2(±2)×104, 1.76(±1)×105 and 1.5(±2)×103 M−1 respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 °C). The stability of metal ion binding to the sensory site follows the Irving–Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.

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