scholarly journals Identification of Glu-120 as the catalytic nucleophile in Streptomyces lividans endoglucanase CelB

1998 ◽  
Vol 336 (1) ◽  
pp. 139-145 ◽  
Author(s):  
David L. ZECHEL ◽  
Shouming HE ◽  
Claude DUPONT ◽  
Stephen G. WITHERS
Keyword(s):  
Catalytic Domain ◽  
Neutral Loss ◽  
Competitive Inhibitor ◽  
Streptomyces Lividans ◽  
Acid Base ◽  
Anomeric Configuration ◽  
High Concentrations ◽  
Singly Charged ◽  
Kinetics Of ◽  
Enzyme Intermediate

Streptomyces lividans CelB is a family-12 endoglucanase that hydrolyses cellulose with retention of anomeric configuration. A recent X-ray structure of the catalytic domain at 1.75 Å resolution has led to the preliminary assignment of Glu-120 and Glu-203 as the catalytic nucleophile and general acid–base respectively [Sulzenbacher, Shareck, Morosoli, Dupont and Davies (1997) Biochemistry 36, 16032–16039]. The present study confirms the identity of the nucleophile by trapping the glycosyl-enzyme intermediate with the mechanism-based inactivator 2´,4´-dinitrophenyl 2-deoxy-2-fluoro-β-d-cellobioside (2FDNPC). The kinetics of inactivation proceeded in a saturable fashion, yielding the parameters kinact = 0.29±0.02 min-1 and Kinact = 0.72±0.08 mM. Uncompetitive inhibition was observed at high concentrations of 2FDNPC (Ki = 9±1 mM), a behaviour that was also observed with the substrate 2´,4´-dinitrophenyl β-d-cellobioside (kcat = 40±1 s-1, Km = 0.35±0.03 mM, Ki = 24±4 mM). Protection against inactivation was afforded by the competitive inhibitor cellobiose. The electrospray ionization (ESI) mass spectrum of the intact labelled CelB indicated that the inactivator had labelled the enzyme stoichiometrically. Reactivation of the trapped intermediate occurred spontaneously (kH2O = 0.0022 min-1) or via transglycosylation, with cellobiose acting as an acceptor ligand (kreact = 0.024 min-1, Kreact = 54 mM). Digestion of the labelled enzyme by pepsin followed by LC–ESI–tandem MS (MS–MS) operating in neutral loss mode identified a labelled, singly charged peptide of m/z 947.5 Da. Isolation of this peptide by HPLC and subsequent collision-induced fragmentation by ESI–MS–MS produced a daughter-ion spectrum that corresponded to a sequence (QTEIM) containing Glu-120. The nucleophile Glu-120 and the putative acid–base catalyst Glu-203 are conserved in all known family-12 sequences.

scholarly journals Steady-state kinetics of catecholamine transport by chromaffin-granule ‘ghosts’

1974 ◽  
Vol 144 (2) ◽  
pp. 319-325 ◽  
Author(s):  
J H Phillips
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Affinity Constant ◽  
Chromaffin Granule ◽  
Affinity Constants ◽  
Steady State Kinetics ◽  
Low Concentrations ◽  
High Concentrations ◽  
Chromaffin Granule Ghosts ◽  
Kinetics Of

Resealed chromaffin-granule ‘ghosts’ were used to study the steady-state kinetics of catecholamine transport. The pump has a high affinity for (-)-noradrenaline, (-)-adrenaline, tyramine and 5-hydroxytryptamine (serotonin), but a lower affinity for (+)-noradrenaline. The measured rates of incorporation do not conform to Michaelis–Menten kinetics, but affinity constants for the former substrates are in the range 8–18μm. Reserpine is a potent inhibitor. Incorporation as a function of ATP concentration also fails to show simple kinetics; the affinity constant for ATP is deduced to be about 3mm at 1mm-MgCl2. Adenylyl (βγ-methylene)diphosphonate is a competitive inhibitor at low concentrations, but inhibits more strongly at high concentrations. The pump has a transition temperature at 29°C and does not seem to be identical with the Mg2+-stimulated adenosine triphosphatase of chromaffin granules.

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

1968 ◽  
Vol 19 (03/04) ◽  
pp. 364-367 ◽  
Author(s):  
H. C Hemker ◽  
P. W Hemker
Keyword(s):  
Enzyme Kinetics ◽  
Blood Clotting ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

scholarly journals Open Reading Frame sso2387 from the Archaeon Sulfolobus solfataricus Encodes a Polypeptide with Protein-Serine Kinase Activity

2003 ◽  
Vol 185 (11) ◽  
pp. 3436-3445 ◽  
Author(s):  
Brian H. Lower ◽  
Peter J. Kennelly
Keyword(s):  
Protein Kinases ◽  
Catalytic Domain ◽  
Sulfolobus Solfataricus ◽  
Open Reading Frame ◽  
Serine Kinase ◽  
Eukaryotic Protein Kinase ◽  
Reading Frame ◽  
Eukaryotic Protein ◽  
High Concentrations ◽  
Eukaryotic Protein Kinases

ABSTRACT The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the “eukaryotic” protein kinase superfamily. sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme. Autophosphorylation was detected only at temperatures ≥60°C, whereas phosphorylation of exogenous proteins was detectable at 37°C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser548, with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.

scholarly journals Interaction of difluoro-oxaloacetate with aspartate transaminase

1977 ◽  
Vol 161 (2) ◽  
pp. 383-387 ◽  
Author(s):  
P A Briley ◽  
R Eisenthal ◽  
R Harrison ◽  
G D Smith
Keyword(s):  
Steady State ◽  
Competitive Inhibitor ◽  
Aspartate Transaminase ◽  
Steady State Kinetic ◽  
High Concentrations ◽  
State Kinetic ◽  
Uncompetitive Inhibitor

Diffluoro-oxaloacetate behaves as a competitive inhibitor of 2-oxoglutarate and as an uncompetitive inhibitor with respect to aspartate in steady-state kinetic experiments with cytoplasmic aspartate transaminase. In the presence of high concentrations of aspartate transaminase, difluoro-oxaloacetate is slowly transaminated to difluoro-aspartate, suggesting its use as a kinetic probe to study the reactions of the aminic form of the enzyme.

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 Novel Substrates for Kinases Involved in the Biosynthesis of Inositol Pyrophosphates and Their Enhancement of ATPase Activity of a Kinase

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3601
Author(s):  
Raja Mohanrao ◽  
Ruth Manorama ◽  
Shubhra Ganguli ◽  
Mithun C. Madhusudhanan ◽  
Rashna Bhandari ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Atpase Activity ◽  
Catalytic Domain ◽  
Inositol Phosphate ◽  
Substrate Recognition ◽  
Competitive Inhibitor ◽  
Inositol Polyphosphates ◽  
Inositol Pyrophosphate ◽  
Phosphate Donor ◽  
Inositol Pyrophosphates

IP6K and PPIP5K are two kinases involved in the synthesis of inositol pyrophosphates. Synthetic analogs or mimics are necessary to understand the substrate specificity of these enzymes and to find molecules that can alter inositol pyrophosphate synthesis. In this context, we synthesized four scyllo-inositol polyphosphates—scyllo-IP5, scyllo-IP6, scyllo-IP7 and Bz-scyllo-IP5—from myo-inositol and studied their activity as substrates for mouse IP6K1 and the catalytic domain of VIP1, the budding yeast variant of PPIP5K. We incubated these scyllo-inositol polyphosphates with these kinases and ATP as the phosphate donor. We tracked enzyme activity by measuring the amount of radiolabeled scyllo-inositol pyrophosphate product formed and the amount of ATP consumed. All scyllo-inositol polyphosphates are substrates for both the kinases but they are weaker than the corresponding myo-inositol phosphate. Our study reveals the importance of axial-hydroxyl/phosphate for IP6K1 substrate recognition. We found that all these derivatives enhance the ATPase activity of VIP1. We found very weak ligand-induced ATPase activity for IP6K1. Benzoyl-scyllo-IP5 was the most potent ligand to induce IP6K1 ATPase activity despite being a weak substrate. This compound could have potential as a competitive inhibitor.

The Effect of Solvent on the Acid-Base Kinetics of the Excited State of β-Naphthol1

1965 ◽  
Vol 69 (6) ◽  
pp. 2044-2059 ◽  
Author(s):  
Norman M. Trieff ◽  
Benson R. Sundheim
Keyword(s):  
Excited State ◽  
Acid Base ◽  
Effect Of Solvent ◽  
Kinetics Of

scholarly journals Kinetics of acid-base parameters in conventional hemodialysis

2019 ◽  
Vol 52 (1) ◽  
Author(s):  
J.R. Lugon ◽  
G.R.M. Pereira ◽  
J.P. Strogoff-de-Matos ◽  
A.J. Peixoto
Keyword(s):  
Acid Base ◽  
Base Parameters ◽  
Kinetics Of ◽  
Conventional Hemodialysis

scholarly journals Evaluation of the kinetics of oxidation and removal of organic matter in the self-purification of a mountain river

DYNA ◽  
2015 ◽  
Vol 82 (191) ◽  
pp. 183-193 ◽  
Author(s):  
Jorge Virgilio Rivera Gutiérrez
Keyword(s):  
Water Quality ◽  
Water System ◽  
The Self ◽  
Organic Load ◽  
Mountain River ◽  
Kinetics Of Oxidation ◽  
Kinetic Rates ◽  
High Concentrations ◽  
Kinetics Of

The study is based on the determination of the kinetic rates and assessment of self-purification of the Frio River, due to the uptake of organic load. The kinetic rates were calculated by applying differential and logarithmic methods on concentrations of water quality determinants present in each of the (7) reach of the river. The water system easily recovers the amount of oxygen, k<sub>d</sub>= 0.4, k<sub>a</sub> 3.2 d<sup>-1</sup>, only receives 27.7 Ton. d<sup>-1</sup>, the organic load, making high concentrations of carbon, ammonium and remain sediment. The length Influence of discharges, LIV- BOD yielded a mean per tranche of 10 km, compared to 3 km each way, means that the river can´t self- purification that need more length of travel. The study illustrates the modeling of the determinants of quality, developed by the QUAL2K, using the calculated rates.

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