scholarly journals Specific Sites in the Cytoplasmic N Terminus Modulate Conformational Transitions of the Na,K-ATPase

2007 ◽  
Vol 282 (46) ◽  
pp. 33691-33697 ◽  
Author(s):  
Rosemarie Scanzano ◽  
Laura Segall ◽  
Rhoda Blostein
Keyword(s):  
Steady State ◽  
Conformational Equilibrium ◽  
Conformational Transitions ◽  
Catalytic Cycle ◽  
Flexible Structure ◽  
Atp Binding ◽  
Deletion Mutagenesis ◽  
N Terminus

The cytoplasmic N terminus of the Na,K-ATPase is a highly charged and flexible structure that comprises three predicted helical regions including H1 spanning residues 27 to 33 and H2 spanning residues 42 to 50. Previous deletion mutagenesis experiments showed that deletion of residues up to and including most of H2 shifts the E1/E2 conformational equilibrium toward E1. The present study describes a clustered charge-to-alanine mutagenesis approach designed to delineate specific sites within the N terminus that modulate the steady-state E1 ↔ E2 and E1P ↔ E2P poise. Criteria to assess shifts in poise include (i) sensitivity to inhibition by inorganic orthovanadate to assess overall poise; (ii) K+-sensitivity of Na-ATPase measured at micromolar ATP to assess changes in the E2(K) + ATP → E1·ATP + K+ rate; (iii) K′ATP for low-affinity ATP binding at the latter step; (iv) overall catalytic turnover, and (v) the E1P → E2P transition. The results of alanine replacements in H1 (31KKE) suggest that this site stabilizes E2P and to a lesser extent E2. In H2, residues within 47HRK have a role in stabilizing E2 but not E2P as revealed with double mutants 31KKE → AAA/47H → A and 31KKE → AAA/47HRK → AAA. Taken together, these observations suggest that sites 31KKE in H1 and 47HRK in H2 have distinct roles in modulating the enzyme's conformational transitions during the catalytic cycle of the enzyme.

scholarly journals A novel approach to distinguish between enzyme mechanisms: quasi-steady-state kinetic analysis of the prostaglandin H synthase peroxidase reaction

2003 ◽  
Vol 372 (3) ◽  
pp. 713-724 ◽  
Author(s):  
Peter V. VRZHESHCH ◽  
Elena A. BATANOVA ◽  
Alevtina T. MEVKH ◽  
Sergei D. VARFOLOMEEV ◽  
Irina G. GAZARYAN ◽  
...  
Keyword(s):  
Experimental Data ◽  
Steady State ◽  
Catalytic Cycle ◽  
Prostaglandin H Synthase ◽  
Compound I ◽  
Novel Approach ◽  
Steady State Kinetic ◽  
Enzyme Intermediates ◽  
Prostaglandin H ◽  
State Kinetic

A method of analysis for steady-state kinetic data has been developed that allows relationships between key partial reactions in the catalytic cycle of a functioning enzyme to be determined. The novel approach is based on a concept of scalar and vector ‘kinetic connectivities’ between enzyme intermediates in an arbitrary enzyme mechanism. The criterion for the agreement between experimental data and a proposed kinetic model is formulated as the kinetic connectivity of intermediate forms of the enzyme. This concept has advantages over conventional approaches and is better able to describe the complex kinetic behaviour of prostaglandin H synthase (PGHS) when catalysing the oxidation of adrenaline by H2O2. To interpret the experimental data for PGHS, a generalized model for multi-substrate enzyme reactions was developed with provision for irreversible enzyme inactivation. This model showed that two enzyme intermediates must undergo inactivation during the catalytic cycle. These forms are proposed to be PGHS compound I and a compound I–adrenaline complex.

scholarly journals The flexible N-terminus of BchL protects its [4Fe-4S] cluster in oxygenic environments and autoinhibits activity

2019 ◽  
Author(s):  
Elliot Corless ◽  
Syed Muhammad Saad Imran ◽  
Maxwell B. Watkins ◽  
Sofia Origanti ◽  
John-Paul Bacik ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Enzyme Stability ◽  
Binding Pocket ◽  
Free Form ◽  
Release Mechanism ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Substrate Reduction ◽  
Evolutionarily Conserved ◽  
N Terminus

AbstractThe dark-operative protochlorophyllide oxidoreductase (DPOR) enzyme contains two [4Fe-4S]- containing component proteins (BchL and BchNB) that assemble in an ATP-dependent fashion to coordinate electron transfer and reduction of protochlorophyllide to chlorophyllide. Photosynthesis generates an oxygenic environment that is non-optimal for [Fe-S] clusters and we here present an elegant evolutionarily conserved mechanism in BchL to protect its [4Fe-4S] cluster. We present a crystal structure of BchL in the nucleotide-free form with an ordered N-terminus that shields the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions that perturb the shielding of the [4Fe-4S] cluster produce an unstable, but hyper-active enzyme complex, suggesting a role for the N-terminus in both auto-inhibition and enzyme stability. Upon ATP binding, a patch of amino acids, Asp-Phe-Asp (‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits and relieves the auto-inhibition by the N-terminus. A linked BchL dimer with one defective ATP-binding site does not support substrate reduction, illustrating that nucleotide binding to both subunits is a prerequisite for the inter-subunit cross stabilization. We propose that ATP-binding produces a conformational compaction of the BchL homodimer leading to a release of the flexible N-terminus from blocking the [4Fe-4S] cluster and promotes complex formation with BchNB to drive electron transfer. The auto-inhibitive feature and release mechanism appear unique to DPOR and is not found in the structurally similar nitrogenase.

Broadband Dynamic Modification Using Feedforward Control

1995 ◽  
Author(s):  
Thomas E. Alberts ◽  
Hemanshu R. Pota
Keyword(s):  
Steady State ◽  
Feedforward Control ◽  
Design Procedure ◽  
Harmonic Excitation ◽  
Flexible Structure ◽  
Minimum Phase ◽  
Original Result ◽  
Modal Expansion ◽  
General Proof ◽  
Dynamic Modification

Abstract This paper presents a general proof of a result due to Fuller and Burdisso, that asserts that system eigenvalues can be modified using feedforward control. The original result applies to the case of steady-state harmonic excitation. This paper extends that work to allow for broadband excitation. The results apply to any flexible structure representable using modal expansion, and are applicable to systems with non-minimum phase zeros. A design procedure is presented to allow arbitrary assignment of the controlled system’s poles, using a fixed feedforward compensator.

Abstract 898: Probing accessibility and conformational changes in ATP-binding sites during the catalytic cycle of human P-glycoprotein (ABCB1).

2013 ◽  
Author(s):  
Hong-May Sim ◽  
Jaya Bhatnagar ◽  
Samantha A. Green ◽  
Adam Gonzalez ◽  
Eduardo E. Chufan ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Catalytic Cycle ◽  
Atp Binding ◽  
P Glycoprotein

scholarly journals Pre-steady-state kinetic study of the effects of K+ on the partial reactions of the catalytic cycle of the plasma membrane Ca2+-ATPase

1996 ◽  
Vol 315 (2) ◽  
pp. 673-677 ◽  
Author(s):  
Claudio J. HERSCHER ◽  
Alcides F. REGA ◽  
Hugo P. ADAMO
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Atp Hydrolysis ◽  
State Level ◽  
Red Cells ◽  
Catalytic Cycle ◽  
Steady State Kinetic ◽  
Phosphorylation Reaction ◽  
Apparent Concentration ◽  
Hydrolysis Of

The effects of 100 mM K+ on the partial reactions that take place during ATP hydrolysis by the calcium ion-dependent ATPase from plasma membrane (PM-Ca2+-ATPase) were studied at 37 °C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. At 10 μM [γ-32P]ATP plus non-limiting concentrations of Ca2+ and Mg2+, K+ increased the kapp of formation by 140% to 84±11 s-1 and the steady-state level of phosphoenzyme (EP) by 25% to 3.4±0.17 pmol/mg of protein. If added together with [γ-32P]ATP at the beginning of phosphorylation, K+ was much less effective than if added earlier, indicating that it did not act on the phosphorylation reaction. Measurements of the E2 → E1 transition by phosphorylation showed that in medium with Ca2+ and Mg2+, K+ increased the kapp of the transition by 55% to 14±3 s-1 and the apparent concentration of E1 by 45%, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubated with K+. The presence of K+ did not change the slow decay of EP without Mg2+ but activated the decay of EP made with Mg2+, increasing its kapp by 60% to 91±12 s-1. In contrast with observations made during phosphorylation, if added at the beginning of dephosphorylation K+ was fully effective in favouring decomposition of EP made in medium containing no K+. In the presence of either 3 mM ATP or 3 mM ATP plus calmodulin, which activate hydrolysis of CaE2P, the effect of K+ on dephosphorylation was conserved. Because the sites for K+ are intracellular and the concentration of K+ in normal red cells is above 100 mM, the effects described here must be taken into account to describe the catalytic cycle of the PM-Ca2+-ATPase under physiological conditions.

Pre-steady state transport by erythrocyte band 3 protein: uphill countertransport induced by the impermeant inhibitor H2DIDS

1998 ◽  
Vol 76 (5) ◽  
pp. 807-813 ◽  
Author(s):  
Michael L Jennings ◽  
Jonathan Whitlock ◽  
Anjali Shinde
Keyword(s):  
Steady State ◽  
Anion Exchange ◽  
Sodium Phosphate ◽  
Band 3 ◽  
Catalytic Cycle ◽  
Band 3 Protein ◽  
Molar Excess ◽  
Transport Inhibitor ◽  
Ping Pong

Pre-steady state Cl- efflux experiments have been performed to test directly the idea that the transport inhibitor H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) binds preferentially to the outward-facing state of the transporter. Cells were equilibrated with a medium consisting of 150 mM sodium phosphate, pH 6.2, N2 atmosphere, and 80-250 µM 36Cl-. Addition of H2DIDS (10-fold molar excess compared with band 3) induces a transient efflux of Cl-, as expected if H2DIDS binds more tightly to outward-facing than to inward-facing states. The size of the H2DIDS-induced efflux depends on the Cl- concentration and is about 700 000 ions per cell at the highest concentrations tested. The size of the transient efflux is larger than would be expected if the catalytic cycle for anion exchange involved one pair of exchanging anions per band 3 dimer. These results are completely consistent with a ping-pong mechanism of anion exchange in which the catalytic cycle consists of one pair of exchanging anions per subunit of the band 3 dimer.Key words: anion exchange, erythrocyte, stilbenedisulfonate, chloride.

scholarly journals Changes in Steady-state Conformational Equilibrium Resulting from Cytoplasmic Mutations of the Na,K-ATPase α-Subunit

1998 ◽  
Vol 273 (36) ◽  
pp. 23086-23092 ◽  
Author(s):  
Nanna Boxenbaum ◽  
Stewart E. Daly ◽  
Zahid Z. Javaid ◽  
Lois K. Lane ◽  
Rhoda Blostein
Keyword(s):  
Steady State ◽  
Conformational Equilibrium ◽  
Α Subunit
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