Integration of a Random‐Rapid‐Equilibrium Binding Model of NOX2 Assembly with a Five‐State Rate‐Limiting Model of NOX2 Electron Flow facilitating Superoxide Production

Control analysis is used to analyse and quantify the concept of a rate-limiting step within an enzyme. The extent to which each rate constant within the enzyme limits the steady-state rate of the enzyme and the levels of enzyme intermediate species are quantified as flux and concentration control coefficients. These coefficients are additive and obey summation theorems. The control coefficients of triose phosphate isomerase, carbamate kinase and lactate dehydrogenase are calculated from literature values of the rate constants. It is shown that, contrary to previous assumption, these enzymes do not have a unique rate-limiting step, but rather flux control is shared by several rate constants and varies with substrate, product and effector concentrations, and with the direction of the reaction. Thus the general assumption that an enzyme will have a unique rate-limiting step is unjustified.

Download Full-text

Effect of pyrophosphate ions and alkaline pH on the kinetics of propionaldehyde oxidation by sheep liver cytosolic aldehyde dehydrogenase

Biochemical Journal ◽

10.1042/bj2730691 ◽

1991 ◽

Vol 273 (3) ◽

pp. 691-693 ◽

Cited By ~ 1

Author(s):

J P Hill ◽

P D Buckley ◽

L F Blackwell ◽

R L Motion

Keyword(s):

Steady State ◽

Aldehyde Dehydrogenase ◽

Alkaline Ph ◽

Activation Effect ◽

Ph Values ◽

Sheep Liver ◽

Steady State Rate ◽

State Rate ◽

Rate Limiting ◽

Kinetics Of

Pyrophosphate ions activate the steady-state rate of oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase at alkaline pH values. The steps in the mechanism governing the release of NADH from terminal enzyme. NADH complexes have been shown to be rate-limiting at pH 7.6 [MacGibbon, Buckley & Blackwell (1977) Biochem J. 165, 455-462]. These steps are shown to be also rate-limiting at more alkaline pH values, and it is through an acceleration of these steps that pyrophosphate ions exert their activation effect.

Download Full-text

An equilibrium binding model for CpG DNA-dependent dimerization of Toll-like receptor 9

10.1101/573238 ◽

2019 ◽

Author(s):

Stephanie Reikine ◽

Stephen H. McLaughlin ◽

Christopher M. Johnson ◽

Yorgo Modis

Keyword(s):

Light Scattering ◽

Inflammatory Response ◽

Innate Immune ◽

Binding Parameters ◽

Toll Like Receptor ◽

Binding Model ◽

Cpg Dna ◽

Extracellular Milieu ◽

Equilibrium Binding ◽

One Step

AbstractMicrobial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces dimerization of TLR9 and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical equilibrium ligand binding model. Light scattering and fluorescence polarization assays performed with a recombinant TLR9 ectodomain fragment and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters in our binding model. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.

Download Full-text

Characterization of a Baculovirus-Encoded RNA 5′-Triphosphatase

Journal of Virology ◽

10.1128/jvi.72.9.7057-7063.1998 ◽

1998 ◽

Vol 72 (9) ◽

pp. 7057-7063 ◽

Cited By ~ 30

Author(s):

Christian H. Gross ◽

Stewart Shuman

Keyword(s):

Steady State ◽

Product Formation ◽

Rate Limiting Step ◽

Limiting Step ◽

Mrna Capping ◽

Steady State Rate ◽

Rna Triphosphatase ◽

State Rate ◽

Nuclear Polyhedrosis ◽

Rate Limiting

ABSTRACT Autographa californica nuclear polyhedrosis virus (AcNPV) encodes a 168-amino-acid polypeptide that contains the signature motif of the superfamily of protein phosphatases that act via a covalent cysteinyl phosphate intermediate. The sequence of the AcNPV phosphatase is similar to that of the RNA triphosphatase domain of the metazoan cellular mRNA capping enzyme. Here, we show that the purified recombinant AcNPV protein is an RNA 5′-triphosphatase that hydrolyzes the γ-phosphate of triphosphate-terminated poly(A); it also hydrolyzes ATP to ADP and GTP to GDP. The phosphatase sediments as two discrete components in a glycerol gradient: a 9.5S oligomer and 2.5S putative monomer. The 2.5S form of the enzyme releases 32Pi from 1 μM γ-32P-labeled triphosphate-terminated poly(A) with a turnover number of 52 min−1 and converts ATP to ADP with V max of 8 min−1and Km of 25 μM ATP. The 9.5S oligomeric form of the enzyme displays an initial pre-steady-state burst of ADP and Pi formation, which is proportional to and stoichiometric with the enzyme, followed by a slower steady-state rate of product formation (approximately 1/10 of the steady-state rate of the 2.5S enzyme). We surmise that the oligomeric enzyme is subject to a rate-limiting step other than reaction chemistry and that this step is either distinct from or slower than the rate-limiting step for the 2.5S enzyme. Replacing the presumptive active site nucleophile Cys-119 by alanine abrogates RNA triphosphatase and ATPase activity. Our findings raise the possibility that baculoviruses encode enzymes that cap the 5′ ends of viral transcripts synthesized at late times postinfection by a virus-encoded RNA polymerase.

Download Full-text

Comparison of promoter-specific events during transcription initiation in mycobacteria

Microbiology ◽

10.1099/mic.0.038620-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 1942-1952 ◽

Cited By ~ 18

Author(s):

Arnab China ◽

Priyanka Tare ◽

Valakunja Nagaraja

Keyword(s):

Protein Interactions ◽

Transcription Initiation ◽

Kinetic Properties ◽

Kinetic Rate ◽

Rate Limiting Step ◽

Equilibrium Binding ◽

Transcription Process ◽

Kinetic Rate Constants ◽

Rate Limiting ◽

Kinetics Of

DNA–protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter–RNAP interactions during transcription initiation in the σ A-dependent promoters P rrnAPCL1 , P rrnB and P gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

Download Full-text

A procedure for obtaining initial estimates of parameters appearing in steady-state rate or equilibrium binding equations

Canadian Journal of Biochemistry ◽

10.1139/o78-104 ◽

1978 ◽

Vol 56 (7) ◽

pp. 697-701 ◽

Cited By ~ 2

Author(s):

I. G. Darvey ◽

E. J. Walker

Keyword(s):

Steady State ◽

Enzyme Kinetics ◽

Binding Studies ◽

Equilibrium Binding ◽

Initial Substrate ◽

Steady State Rate ◽

State Rate ◽

Using Data ◽

Fitting In ◽

Initial Substrate Concentration

A 'peeling' procedure is described for obtaining initial estimates of the parameters in the equation:[Formula: see text]where P(x) and Q(x) are polynomials in x. The method is illustrated, in the context of enzyme kinetics, using data which are fitted to the following equation:[Formula: see text]where v denotes the initial steady-state velocity at an initial substrate concentration S, and a1, a2, b1 and b2 are non-negative constants. The applicability and limitations of the method for data fitting in fields such as enzyme kinetics and ligand-binding studies are discussed.

Download Full-text

Leishmania pifanoi Amastigotes Avoid Macrophage Production of Superoxide by Inducing Heme Degradation

Infection and Immunity ◽

10.1128/iai.73.12.8322-8333.2005 ◽

2005 ◽

Vol 73 (12) ◽

pp. 8322-8333 ◽

Cited By ~ 48

Author(s):

Nam-Kha Pham ◽

Jennifer Mouriz ◽

Peter E. Kima

Keyword(s):

Nadph Oxidase ◽

Subcellular Fractions ◽

Superoxide Production ◽

Heme Oxygenase 1 ◽

Heme Degradation ◽

Nadph Oxidase Complex ◽

Immature Form ◽

Oxidase Enzyme ◽

Infected Cells ◽

Rate Limiting

ABSTRACT Whereas infections of macrophages by promastigote forms of Leishmania mexicana pifanoi induce the production of superoxide, infections by amastigotes barely induce superoxide production. Several approaches were employed to gain insight into the mechanism by which amastigotes avoid eliciting superoxide production. First, in experiments with nitroblue tetrazolium, we found that 25% of parasitophorous vacuoles (PVs) that harbor promastigotes are positive for the NADPH oxidase complex, in contrast to only 2% of PVs that harbor amastigotes. Second, confocal microscope analyses of infected cells labeled with antibodies to gp91phox revealed that this enzyme subunit is found in PVs that harbor amastigotes. Third, in immunoblots of subcellular fractions enriched with PVs from amastigote-infected cells and probed with antibodies to gp91phox, only the 65-kDa premature form of gp91phox was found. In contrast, subcellular fractions from macrophages that ingested zymosan particles contained both the 91- and 65-kDa forms of gp91phox. This suggested that only the immature form of gp91phox is recruited to PVs that harbor amastigotes. Given that gp91phox maturation is dependent on the availability of heme, we found that infections by Leishmania parasites induce an increase in heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation. Infections by amastigotes performed in the presence of metalloporphyrins, which are inhibitors of HO-1, resulted in superoxide production by infected macrophages. Taken together, we propose that Leishmania amastigotes avoid superoxide production by inducing an increase in heme degradation, which results in blockage of the maturation of gp91phox, which prevents assembly of the NADPH oxidase enzyme complex.

Download Full-text

A novel PSII photosynthetic control is activated in anoxic cultures of green algae

10.1101/2021.12.01.470728 ◽

2021 ◽

Author(s):

Yuval Milrad ◽

Valeria Nagy ◽

Szilvia Toth ◽

Iftach Yacoby

Keyword(s):

Green Algae ◽

Electron Flow ◽

Spectroscopic Techniques ◽

Rate Limiting Step ◽

Fluctuating Light ◽

Photosynthetic Complexes ◽

Photosynthetic Control ◽

Control Set ◽

Photosynthetic Regulation ◽

Rate Limiting

Photosynthetic green algae face an ever-changing environment of fluctuating light as well as unstable oxygen levels, which via the production of free radicals constantly challenges the integrity of the photosynthetic complexes. To face such challenges, a complex photosynthetic control network monitors and tightly control the membrane redox potential. Here, we show that not only that the photosynthetic control set the rate limiting step of photosynthetic linear electron flow, but also, upon its ultimate dissipation, it triggers intrinsic alternations in the activity of the photosynthetic complexes. These changes have a grave and prolonged effect on the activity of photosystem II, leading to a massive 3-fold decrease in its electron output. We came into this conclusion via studying a variety of green algae species and applying advance mass-spectrometry and diverse spectroscopic techniques. Our results shed new light on the mechanism of photosynthetic regulation and provide new target for improving photosynthesis.

Download Full-text