scholarly journals Laser Photolysis Investigations of Ligand Binding With Models of the Active Site of Respiratory Hemoproteins: Kinetic and Thermodynamic Aspects

1990 ◽  
Vol 10 (5-6) ◽  
pp. 297-318 ◽  
Author(s):  
D. Lavalette ◽  
C. Tetreau ◽  
M. Momenteau ◽  
J. Mispelter ◽  
J. M. Lhoste ◽  
...  
Keyword(s):  
Active Site ◽  
Structural Parameters ◽  
Dissociation Rate ◽  
Laser Photolysis ◽  
Concentration Jump ◽  
Linear Free Energy ◽  
Energy Relationships ◽  
Dissociation Rate Constants ◽  
Kinetics Of

During the past 15 years, laser photolysis has been the method of choice for probing the complex reaction kinetics of respiratory proteins. In an attempt to determine the structural parameters which govern their reactivity, synthetic heme model compounds capable of simulating particular aspects of the reactivity of the active site of hemoproteins have been successively proposed. Laser photolysis of heme compounds merely induces a reversible photodissociation of one ligand at a time. This is equivalent to performing a fast concentration jump "in situ" and provides a powerful, fast and "clean" chemical relaxation technique. To gather association and dissociation rate constants of various ligands (O2, CO, nitrogenous bases) special methods have been developed or adapted. The problem of comparing and classifying a large number of collected data has been greatly simplified by introducing a Linear Free Energy Relationships formalism. In the first part of this paper, some of the methods and concepts which have emerged from several years of investigations of heme proteins and heme models and which are of a sufficient generality to be useful in other fields of chemical kinetics are reviewed. In the second part of the paper we present the application of the preceding methods to a kinetic study of a series of heme models which were specifically designed to investigate the important problem of H-bonding as a stabilizing factor of the oxygenated heme model and hemoprotein complexes.

scholarly journals Desensitization Contributes to the Synaptic Response of Gain-of-Function Mutants of the Muscle Nicotinic Receptor

2006 ◽  
Vol 128 (5) ◽  
pp. 615-627 ◽  
Author(s):  
Sergio Elenes ◽  
Ying Ni ◽  
Gisela D. Cymes ◽  
Claudio Grosman
Keyword(s):  
Nicotinic Receptor ◽  
Dissociation Rate ◽  
Gain Of Function ◽  
Naturally Occurring ◽  
Channel Opening ◽  
Speed Up ◽  
High Concentrations ◽  
Dissociation Rate Constants ◽  
Kinetics Of ◽  
Peak Value

Although the muscle nicotinic receptor (AChR) desensitizes almost completely in the steady presence of high concentrations of acetylcholine (ACh), it is well established that AChRs do not accumulate in desensitized states under normal physiological conditions of neurotransmitter release and clearance. Quantitative considerations in the framework of plausible kinetic schemes, however, lead us to predict that mutations that speed up channel opening, slow down channel closure, and/or slow down the dissociation of neurotransmitter (i.e., gain-of-function mutations) increase the extent to which AChRs desensitize upon ACh removal. In this paper, we confirm this prediction by applying high-frequency trains of brief (∼1 ms) ACh pulses to outside-out membrane patches expressing either lab-engineered or naturally occurring (disease-causing) gain-of-function mutants. Entry into desensitization was evident in our experiments as a frequency-dependent depression in the peak value of succesive macroscopic current responses, in a manner that is remarkably consistent with the theoretical expectation. We conclude that the comparatively small depression of the macroscopic currents observed upon repetitive stimulation of the wild-type AChR is due, not to desensitization being exceedingly slow but, rather, to the particular balance between gating, entry into desensitization, and ACh dissociation rate constants. Disruption of this fine balance by, for example, mutations can lead to enhanced desensitization even if the kinetics of entry into, and recovery from, desensitization themselves are not affected. It follows that accounting for the (usually overlooked) desensitization phenomenon is essential for the correct interpretation of mutagenesis-driven structure–function relationships and for the understanding of pathological synaptic transmission at the vertebrate neuromuscular junction.

scholarly journals Oversized galactosides as a probe for conformational dynamics in LacY

2018 ◽  
Vol 115 (16) ◽  
pp. 4146-4151 ◽  
Author(s):  
Irina Smirnova ◽  
Vladimir Kasho ◽  
Xiaoxu Jiang ◽  
Hong-Ming Chen ◽  
Stephen G. Withers ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Dynamics ◽  
Dissociation Rate ◽  
Binding Kinetics ◽  
Lactose Permease ◽  
E Coli ◽  
Open Conformation ◽  
Dissociation Rate Constants ◽  
Kinetics Of ◽  
Micromolar Range

Binding kinetics of α-galactopyranoside homologs with fluorescent aglycones of different sizes and shapes were determined with the lactose permease (LacY) of Escherichia coli by FRET from Trp151 in the binding site of LacY to the fluorophores. Fast binding was observed with LacY stabilized in an outward-open conformation (kon = 4–20 μM−1·s−1), indicating unobstructed access to the binding site even for ligands that are much larger than lactose. Dissociation rate constants (koff) increase with the size of the aglycone so that Kd values also increase but remain in the micromolar range for each homolog. Phe27 (helix I) forms an apparent constriction in the pathway for sugar by protruding into the periplasmic cavity. However, replacement of Phe27 with a bulkier Trp does not create an obstacle in the pathway even for large ligands, since binding kinetics remain unchanged. High accessibility of the binding site is also observed in a LacY/nanobody complex with partially blocked periplasmic opening. Remarkably, E. coli expressing WT LacY catalyzes transport of α- or β-galactopyranosides with oversized aglycones such as bodipy or Aldol518, which may require an extra space within the occluded intermediate. The results confirm that LacY specificity is strictly directed toward the galactopyranoside ring and also clearly indicate that the opening on the periplasmic side is sufficiently wide to accommodate the large galactoside derivatives tested here. We conclude that the actual pathway for the substrate entering from the periplasmic side is wider than the pore diameter calculated in the periplasmic-open X-ray structures.

The Kinetic Activation Volumes for the Binding of Iodide to Cobalamin (Vitamin B12) Studied on a High Pressure Laser Temperature Jump Apparatus

1974 ◽  
Vol 52 (6) ◽  
pp. 910-914 ◽  
Author(s):  
Brian B. Hasinoff
Keyword(s):  
High Pressure ◽  
Metal Ion ◽  
Temperature Jump ◽  
Outer Sphere ◽  
Dissociation Rate ◽  
Vitamin B ◽  
Laser Temperature Jump ◽  
Dissociation Rate Constants ◽  
Kinetics Of ◽  
Activated Complex

The relaxation kinetics of the reaction of iodide with cobalamin (vitamin B12) were studied on a high pressure laser temperature jump apparatus in aqueous solution at 25° and ionic strength 0.2 M. Analysis of the pressure dependence of the formation and dissociation rate constants gave their respective volumes of activation to be: ΔVf* = 5.50 ± 0.8 cm3 mol−1 and ΔVd* = 11.5 ± 1.6 cm3 mol−1 The positive activation volume for formation of the complex, ΔVf*, after appropriate correction for the volume change due to formation of an outer sphere complex, is consistent with a dissociative type mechanism in which the metal ion – water bond is stretched in the activated complex.

scholarly journals Transition State Interactions in a Promiscuous Enzyme: Sulfate and Phosphate Monoester Hydrolysis byPseudomonas aeruginosaArylsulfatase

2018 ◽  
Author(s):  
Bert van Loo ◽  
Ryan Berry ◽  
Usa Boonyuen ◽  
Mark F. Mohamed ◽  
Marko Golicnik ◽  
...  
Keyword(s):  
Active Site ◽  
Isotope Effects ◽  
Charge Compensation ◽  
Linear Free Energy ◽  
Uncatalyzed Reaction ◽  
Leaving Group ◽  
Energy Relationships ◽  
Increased Sensitivity ◽  
Leaving Group Ability ◽  
Active Site Mutants

ABSTRACTPseudomonas aeruginosaarylsulfatase (PAS) hydrolyses sulfate and, promiscuously, phosphate monoesters. Enzyme-catalyzed sulfate transfer is crucial to a wide variety of biological processes, but detailed studies of the mechanistic contributions to its catalysis are lacking. We present an investigation based on linear free energy relationships (LFERs) and kinetic isotope effects (KIEs) of PAS and active site mutants that suggest a key role for leaving group (LG) stabilization. In LFERs wild type PAS has a much less negative Br0nsted coefficient (βleaving groupobs-Enz= −0.33) than the uncatalyzed reaction (βleavingroupobs= −1.81). This situation is diminished when cationic active site groups are exchanged for alanine. The considerable degree of bond breaking during the TS is evidenced by an18ObridgeKIE of 1.0088. LFER and KIE data for several active site mutants point to leaving group stabilization by active-site lysine K375, in cooperation with histidine H211.15N KIEs combined with an increased sensitivity to leaving group ability of the sulfatase activity in neat D2O (Δβleaving groupH-D= +0.06) suggest that the mechanism for S-Obridgebond fission shifts, with decreasing leaving group ability, from charge compensation via Lewis acid interactions towards direct proton donation.18OnonbridgeKIEs indicate that the TS for PAS-catalyzed sulfate monoester hydrolysis has a significantly more associative character compared to the uncatalyzed reaction, while PAS-catalyzed phosphate monoester hydrolysis does not show this shift. This difference in enzyme-catalyzed TSs appears to be the major factor favoring specificity toward sulfate over phosphate in this promiscuous hydrolase, since other features are either too similar (uncatalyzed TS) or inherently favor phosphate (charge).

scholarly journals Sulfide may directly modify cytoplasmic hemoglobin deoxygenation in Solemya reidi gills

1996 ◽  
Vol 199 (6) ◽  
pp. 1343-1352 ◽  
Author(s):  
D Kraus ◽  
J Doeller ◽  
C Powell
Keyword(s):  
Oxygen Consumption ◽  
Hydrogen Sulfide ◽  
Intracellular Ph ◽  
Oxygen Consumption Rate ◽  
Optical Spectrum ◽  
Consumption Rate ◽  
Dissociation Rate ◽  
High Concentration ◽  
Dissociation Rate Constants

The clam Solemya reidi, which survives in sulfide-rich sediments, houses intracellular sulfide-oxidizing bacteria as symbionts in its gills. The gill bacteriocytes also contain a high concentration of cytoplasmic hemoglobin. Although the in situ hemoglobin optical spectrum was not altered in the presence of hydrogen sulfide, hemoglobin deoxygenation was significantly slowed and incomplete when sulfide was present. A sulfide-mediated decrease in oxygen consumption rate, a shift in intracellular pH or the conversion of hemoglobin to an unusual derivative could all slow in situ hemoglobin deoxygenation. However, under low sulfide levels at which deoxygenation is incomplete, oxygen consumption rate was not inhibited, intracellular pH decreased by less than 0.1 units and the only hemoglobin derivatives present were deoxyhemoglobin and oxyhemoglobin. These results and preliminary measurements of the isolated gill hemoglobin dissociation rate constants suggest that sulfide or a rapidly formed oxidation product may directly influence the rate of Solemya reidi gill hemoglobin deoxygenation.

scholarly journals The Essential Functional Interplay of the Catalytic Groups in Acid Phosphatase

2021 ◽  
Author(s):  
Martin Pfeiffer ◽  
Bernd Nidetzky ◽  
Rory Crean ◽  
Cátia Moreira ◽  
Antonietta Parracino ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Active Site ◽  
Enzymatic Reaction ◽  
Valence Bond ◽  
Phosphoryl Transfer ◽  
Enzyme Design ◽  
Linear Free Energy ◽  
Mechanistic Pathway ◽  
Empirical Valence Bond ◽  
Energy Relationships

Cooperative interplay between the functional devices of a preorganized active site is fundamental to enzyme catalysis. A deepened understanding of this phenomenon is central to elucidating the remarkable efficiency of natural enzymes, and provides an essential benchmark for enzyme design and engineering. Here, we study the functional interconnectedness of the catalytic nucleophile (His18) in an acid phosphatase by analyzing the consequences of its replacement with aspartate. We present crystallographic, biochemical and computational evidence for a conserved mechanistic pathway via a phospho-enzyme intermediate on Asp18. Linear free-energy relationships for phosphoryl transfer from phosphomonoester substrates to His18/Asp18 provide evidence for cooperative interplay between the nucleophilic and general-acid catalytic groups in the wildtype enzyme, and its substantial loss in the H18D variant. As an isolated factor of phosphatase efficiency, the advantage of a histidine compared to an aspartate nucleophile is around 10^4-fold. Cooperativity with the catalytic acid adds ≥10^2-fold to that advantage. Empirical valence bond simulations of phosphoryl transfer from glucose 1-phosphate to His and Asp in the enzyme explain the loss of activity of the Asp18 enzyme through a combination of impaired substrate positioning in the Michaelis complex, as well as a shift from early to late protonation of the leaving group in the H18D variant. The evidence presented furthermore suggests that the cooperative nature of catalysis distinguishes the enzymatic reaction from the corresponding reaction in solution and is enabled by the electrostatic preorganization of the active site. Our results reveal sophisticated discrimination in multifunctional catalysis of a highly proficient phosphatase active site.

scholarly journals CD80 (B7-1) Binds Both CD28 and CTLA-4 with a Low Affinity and Very Fast Kinetics

1997 ◽  
Vol 185 (3) ◽  
pp. 393-404 ◽  
Author(s):  
P.  Anton van der Merwe ◽  
Dale L. Bodian ◽  
Susan Daenke ◽  
Peter Linsley ◽  
Simon J. Davis
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
Dissociation Rate ◽  
Fast Kinetics ◽  
Surface Molecules ◽  
Kd Values ◽  
Dissociation Rate Constants ◽  
Antigen Presenting ◽  
Kinetics Of ◽  
Rapid Binding

The structurally related T cell surface molecules CD28 and CTLA-4 interact with cell surface ligands CD80 (B7-1) and CD86 (B7-2) on antigen-presenting cells (APC) and modulate T cell antigen recognition. Preliminary reports have suggested that CD80 binds CTLA-4 and CD28 with affinities (Kd values ∼12 and ∼200 nM, respectively) that are high when compared with other molecular interactions that contribute to T cell–APC recognition. In the present study, we use surface plasmon resonance to measure the affinity and kinetics of CD80 binding to CD28 and CTLA-4. At 37°C, soluble recombinant CD80 bound to CTLA-4 and CD28 with Kd values of 0.42 and 4 μM, respectively. Kinetic analysis indicated that these low affinities were the result of very fast dissociation rate constants (koff); sCD80 dissociated from CD28 and CTLA-4 with koff values of ⩾1.6 and ⩾0.43 s−1, respectively. Such rapid binding kinetics have also been reported for the T cell adhesion molecule CD2 and may be necessary to accommodate dynamic T cell–APC contacts and to facilitate scanning of APC for antigen.

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