Molecular enzymology of the DNA-(adenine-N6)-methyl-transferase M.EcoRV: Kinetic mechanism, kinetics of DNA binding and bending, and linear diffusion

Two discrete furanocoumarin (5- and 8-) O-methyltransferases and a caffeic acid 3-O-methyl-transferase from cell cultures of Ruta graveolens L. have been copurified by affinity chromatography on 1,6-diaminohexane agarose (AH-Sepharose 4B) linked with 5-adenosyl-L-homocysteine (SAH). The furanocoumarin O-methyltransferases, which transfer a methyl group from S-adenosyl-L-methionine (SAM) to the 5- or 8-hydroxyls of linear furanocoumarins, were not retarded by 5-(3-carboxypropanamido)-xanthotoxin (CPAX) immobilized to AH-Sepharose 4B, but addition of SAM to the irrigant buffer led to complete retardation of both enzymes on this affinity system. An analogous phenomenon was observed for the caffeic acid O-methyltransferase, with a ferulic acid ligand coupled to the same insoluble support. SAH was as effective as SAM in promoting binding of the furanocoumarin O-methyltransferases to CPAX and caffeic acid 3-O-methyltransferase to immobilized ferulic acid, respectively. The strong and specific adsorption of these enzymes was abolished by exclusion of SAM or SAH from the irrigant buffer. It is concluded that the enzymes bind first to SAM or SAH, and that this binding process in turn induces the binding site for their specific phenolic substrates or their analogs. Based on these findings, a compulsory–ordered kinetic mechanism for the action of these O-methyltransferases is postulated.

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Oxidation of Olefins Representing Some Structural Units of GR-S

Rubber Chemistry and Technology ◽

10.5254/1.3543370 ◽

1952 ◽

Vol 25 (1) ◽

pp. 21-32 ◽

Cited By ~ 1

Author(s):

W. C. Warner ◽

J. Reid Shelton

Keyword(s):

Phenyl Group ◽

Kinetic Mechanism ◽

Oxidation Reactions ◽

Chain Reaction ◽

Instantaneous Rate ◽

Initial Oxygen ◽

Oxidation Of Olefins ◽

A Minor ◽

The Relationship ◽

Kinetics Of

Abstract Three olefins were oxidized in the liquid phase with molecular oxygen to determine the kinetics of the oxidation reactions and the relationship to oxidation of rubber. The instantaneous rate of oxidation was found to be related to the analytically determined olefin and peroxide concentrations by the equation : Rate=k (unreacted olefin)(peroxide), where rate equals moles of oxygen per mole of original olefin per hour and the parentheses represent molarities. Presence of a phenyl group was found to affect k, but only in a minor way, indicating that the same fundamental kinetic mechanism applies in both aromatic and aliphatic olefins. The data are consistent with the general kinetic mechanism of Bolland involving oxygen attack at the alpha-methylenic group. However, it appears probable that initial oxygen attack can also occur at the double bond, resulting in the formation of a peroxide biradical, which may then react with other olefin molecules, initiating the usual chain reaction mechanism.

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DNA-binding properties of gene-5 protein encoded by bacteriophage M 13. 1. The kinetics of the dissociation of gene-5-protein polynucleotide complexes upon addition of salt

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1988.tb14318.x ◽

1988 ◽

Vol 176 (3) ◽

pp. 589-596 ◽

Cited By ~ 5

Author(s):

Henk BULSINK ◽

Ben J. M. HARMSEN ◽

Cornelis W. HILBERS

Keyword(s):

Dna Binding ◽

Binding Properties ◽

Gene 5 Protein ◽

Dna Binding Properties ◽

Kinetics Of

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Fluorescence Study of DNA Binding and Bending byEcoRI DNA Methyltransferase

The Journal of Physical Chemistry B ◽

10.1021/jp0574293 ◽

2006 ◽

Vol 110 (39) ◽

pp. 19647-19651

Author(s):

Baocheng Ma ◽

Jun Wang ◽

Xiaohong Fang

Keyword(s):

Dna Binding ◽

Dna Methyltransferase ◽

Fluorescence Study ◽

Dna Binding And Bending

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DNA Binding and Bending by the Human Papillomavirus Type 16 E2 Protein

Journal of Biological Chemistry ◽

10.1074/jbc.272.13.8236 ◽

1997 ◽

Vol 272 (13) ◽

pp. 8236-8242 ◽

Cited By ~ 43

Author(s):

Alison Thain ◽

Kenneth Webster ◽

Dave Emery ◽

Anthony R. Clarke ◽

Kevin Gaston

Keyword(s):

Human Papillomavirus ◽

Dna Binding ◽

E2 Protein ◽

Human Papillomavirus Type 16 ◽

Dna Binding And Bending

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The calculation of transcript flux ratios reveals single regulatory mechanisms capable of activation and repression

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1809454115 ◽

2018 ◽

Vol 115 (50) ◽

pp. E11604-E11613 ◽

Cited By ~ 9

Author(s):

Eric A. Galburt

Keyword(s):

Rate Constants ◽

Flux Ratio ◽

Stringent Response ◽

Kinetic Mechanism ◽

Regulation Of Transcription ◽

Sequence Motifs ◽

Response Factors ◽

Initiation Rate ◽

Dna Sequence Motifs ◽

Kinetics Of

The regulation of transcription allows cells to adjust the rate of RNA polymerases (RNAPs) initiated in a promoter-specific manner. Classically, transcription factors are directed to a subset of promoters via the recognition of DNA sequence motifs. However, a unique class of regulators is recruited directly through interactions with RNAP. Surprisingly, these factors may still possess promoter specificity, and it has been postulated that the same kinetic mechanism leads to different regulatory outcomes depending on a promoter’s basal rate constants. However, mechanistic studies of regulation typically report factor activity in terms of changes in the thermodynamics or kinetics of individual steps or states while qualitatively linking these observations to measured changes in transcript production. Here, I present online calculators that allow for the direct testing of mechanistic hypotheses by calculating the steady-state transcript flux in the presence and absence of a factor as a function of initiation rate constants. By evaluating how the flux ratio of a single kinetic mechanism varies across promoter space, quantitative insights into the potential of a mechanism to generate promoter-specific regulatory outcomes are obtained. Using these calculations, I predict that the mycobacterial transcription factor CarD is capable of repression in addition to its known role as an activator of ribosomal genes. In addition, a modification of the mechanism of the stringent response factors DksA/guanosine 5′-diphosphate 3′-diphosphate (ppGpp) is proposed based on their ability to differentially regulate transcription across promoter space. Overall, I conclude that a multifaceted kinetic mechanism is a requirement for differential regulation by this class of factors.

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Real-time kinetic studies of Mycobacterium tuberculosis LexA-DNA interaction

Bioscience Reports ◽

10.1042/bcj20210434 ◽

2021 ◽

Author(s):

Chitral Chatterjee ◽

Soneya Majumdar ◽

Sachin Deshpande ◽

Deepak Pant ◽

Saravanan Matheshwaran

Keyword(s):

Amino Acids ◽

Mycobacterium Tuberculosis ◽

Dna Binding ◽

Kinetic Parameters ◽

Dna Sequences ◽

Kinetic Studies ◽

Dna Interaction ◽

Damage Repair ◽

Inducible Genes ◽

Kinetics Of

Transcriptional repressor, LexA, regulates the “SOS” response, an indispensable bacterial DNA damage repair machinery. Compared to its E.coli ortholog, LexA from Mycobacterium tuberculosis (Mtb) possesses a unique N-terminal extension of additional 24 amino acids in its DNA binding domain (DBD) and 18 amino acids insertion at its hinge region that connects the DBD to the C-terminal dimerization/autoproteolysis domain. Despite the importance of LexA in “SOS” regulation, Mtb LexA remains poorly characterized and the functional importance of its additional amino acids remained elusive. In addition, the lack of data on kinetic parameters of Mtb LexA-DNA interaction prompted us to perform kinetic analyses of Mtb LexA and its deletion variants using Bio-layer Interferometry (BLI). Mtb LexA is seen to bind to different “SOS” boxes, DNA sequences present in the operator regions of damage-inducible genes, with comparable nanomolar affinity. Deletion of 18 amino acids from the linker region is found to affect DNA binding unlike the deletion of the N-terminal stretch of extra 24 amino acids. The conserved RKG motif has been found to be critical for DNA binding. Overall, this study provides insights into the kinetics of the interaction between Mtb LexA and its target “SOS” boxes. The kinetic parameters obtained for DNA binding of Mtb LexA would be instrumental to clearly understand the mechanism of “SOS” regulation and activation in Mtb.

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Simulated growth and microstructure of DyBa2Cu3O7−x with and without Dy2BaCuO5 addition

Journal of Materials Research ◽

10.1557/jmr.1995.0268 ◽

1995 ◽

Vol 10 (2) ◽

pp. 268-273 ◽

Cited By ~ 17

Author(s):

N. Vandewalle ◽

R. Cloots ◽

M. Ausloos

Keyword(s):

Growth Front ◽

Kinetic Mechanism ◽

Optical Observations ◽

Kinetic Processes ◽

Eden Model ◽

Kinetics Of ◽

Simulated Growth ◽

Simple Models ◽

Growth Probability

We present optical observations of magnetically melt-textured DyBa2Cu3O7−x with and without 20 wt. % excess of Dy2BaCuO5. From these observations, we propose some kinetic mechanism of the growth of 123 compounds. Kinetic processes can be simulated on computers. Two (very) simple models derived from the well-known Eden model are presented. They simulate the growth of the grain front. The simulated patterns agree with the observations. The microstructure of such materials cannot be explained by thermodynamic and chemical considerations alone, but explanations must include the kinetics of the growth front as well. From our observations, we conclude that the growth probability ratios g110/g100 and g100/g001 are of the order of 10 and 50, respectively.

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