Kinetic Mechanism of Rat Polymerase β−dsDNA Interactions. Fluorescence Stopped-Flow Analysis of the Cooperative Ligand Binding to a Two-Site One-Dimensional Lattice†

Biochemistry ◽  
2005 ◽  
Vol 44 (4) ◽  
pp. 1251-1267 ◽  
Author(s):  
Roberto Galletto ◽  
Maria J. Jezewska ◽  
Wlodzimierz Bujalowski
Keyword(s):  
Ligand Binding ◽  
Flow Analysis ◽  
Kinetic Mechanism ◽  
Stopped Flow ◽  
Dimensional Lattice ◽  
One Dimensional

scholarly journals Proton release on binding of glutathione to Alpha, Mu and Delta class glutathione transferases

1999 ◽  
Vol 344 (2) ◽  
pp. 419-425 ◽  
Author(s):  
Anna Maria CACCURI ◽  
Giovanni ANTONINI ◽  
Philip G. BOARD ◽  
Michael W. PARKER ◽  
Maria NICOTRA ◽  
...  
Keyword(s):  
Flow Analysis ◽  
Three Dimensional ◽  
Kinetic Mechanism ◽  
Activation Mechanism ◽  
Dimensional Structure ◽  
Binary Complex ◽  
Stopped Flow ◽  
Binding Mechanism ◽  
Proton Extrusion ◽  
Evolutionary Pathway

Potentiometric, spectroscopic and stopped-flow experiments have been performed to dissect the binding mechanism of GSH to selected glutathione S-transferases (GSTs), A1-1, M2-2 and Lucilia cuprina GST, belonging to Alpha, Mu and Delta classes respectively. Both Alpha and Mu isoenzymes quantitatively release the thiol proton of the substrate when the binary complex is formed. Proton extrusion, quenching of intrinsic fluorescence and thiolate formation, diagnostic of different steps along the binding pathway, have been monitored by stopped-flow analysis. Kinetic data are consistent with a multi-step binding mechanism: the substrate is initially bound to form an un-ionized pre-complex [k1⩾ (2-5)×106 M-1˙s-1], which is slowly converted into the final Michaelis complex (k2 = 1100-1200 s-1). Ionization of GSH, fluorescence quenching and proton extrusion are fast events that occur either synchronously or rapidly after the final complex formation. The Delta isoenzyme shows an interesting difference: proton extrusion is almost stoichiometric with thiolate formed at the active site only up to pH 7.0. Above this pH, at least one protein residue acts as internal base to neutralize the thiol proton. These results suggest that the Alpha and Mu enzymes retain not only a similar catalytic outcome and overall three-dimensional structure but also share a similar kinetic mechanism for GSH binding. The Delta GST, which is closely related to the mammalian Theta class enzymes and is distantly related to Alpha and Mu GSTs in the evolutionary pathway, might display a different activation mechanism for GSH.

scholarly journals DIFFERENTIAL EQUATION APPROACH FOR ONE- AND TWO-DIMENSIONAL LATTICE GREEN'S FUNCTION

2007 ◽  
Vol 21 (02n03) ◽  
pp. 139-154 ◽  
Author(s):  
J. H. ASAD
Keyword(s):  
Differential Equation ◽  
Green’S Function ◽  
Recurrence Relation ◽  
Green's Function ◽  
Two Dimensional ◽  
Dimensional Lattice ◽  
One Dimensional ◽  
The One ◽  
Lattice Green's Function ◽  
Lattice Green’S Function

A first-order differential equation of Green's function, at the origin G(0), for the one-dimensional lattice is derived by simple recurrence relation. Green's function at site (m) is then calculated in terms of G(0). A simple recurrence relation connecting the lattice Green's function at the site (m, n) and the first derivative of the lattice Green's function at the site (m ± 1, n) is presented for the two-dimensional lattice, a differential equation of second order in G(0, 0) is obtained. By making use of the latter recurrence relation, lattice Green's function at an arbitrary site is obtained in closed form. Finally, the phase shift and scattering cross-section are evaluated analytically and numerically for one- and two-impurities.

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