Nonlinear steady-state kinetics of chloramphenicol acetyltransferase

1991 ◽  
Vol 69 (9) ◽  
pp. 630-634
Author(s):  
M. James C. Crabbe ◽  
Derek Goode
Keyword(s):  
Steady State ◽  
Chloramphenicol Acetyltransferase ◽  
Binding Region ◽  
Medium Effects ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Kinetics Of ◽  
State Kinetic

Steady-state kinetic analysis of chloramphenicol acetyltransferase showed that medium effects (higher temperatures or pH, higher ionic strengths, or lower values for dielectric constant) altered the kinetic behaviour of the enzyme with acetyl-CoA as substrate, but did not significantly affect behaviour with chloramphenicol. This was manifest as an increase in the degree of the rate equation to a 2:2 function. This is interpreted in terms of perturbations to the enzyme at or near the acetyl-CoA binding region of the enzyme.Key words: acetyl coenzyme A, chloramphenicol, antibiotics, enzyme kinetics.

The Steady State Kinetics of Plasmin and Trypsin Catalysed Hydrolysis of a Number of Tripeptideanilides

1979 ◽  
Author(s):  
U. Christensen ◽  
H-H. Ipsen
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Amino Group ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Ph Range ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
State Kinetic

The steady state kinetic parameters of plasmin and trypsin catalysed hydrolysis of Bz-L-Phe-Val-Arg-pNA, L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA, D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA in the pH-range 6-9 are presented. Ionization of catalytically essential enzymic groups accounts satisfactorily for the pH-dependencies of the kinetic parameters for plas-rain and trypsin reactions with Bz-L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA. The protonation of the α-amino group of L-Phe-Val-Arg-pNA and D-Phe-Val-Arg-pNA (pK=7.0) show some additional effect. The values of the catalytic constants for plasmin and trypsin reactions with these p-nitroanilides are alike those normally found for specific ester substrates, indicating that the deacylation steps are rate determining.

scholarly journals Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases

2012 ◽  
Vol 287 (42) ◽  
pp. 35516-35526 ◽  
Author(s):  
Linlin Zhao ◽  
Matthew G. Pence ◽  
Plamen P. Christov ◽  
Zdzislaw Wawrzak ◽  
Jeong-Yun Choi ◽  
...  
Keyword(s):  
Steady State ◽  
Crystal Structures ◽  
Dna Polymerases ◽  
Base Pairing ◽  
Mutagenic Potential ◽  
Human Dna ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Y Family ◽  
State Kinetic

N2,3-Ethenoguanine (N2,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N2,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N2,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N2,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N2,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N2,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N2,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N2,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N2,3-ϵG.

The mechanistic study of Leishmania major dihydro-orotate dehydrogenase based on steady- and pre-steady-state kinetic analysis

2016 ◽  
Vol 473 (5) ◽  
pp. 651-660 ◽  
Author(s):  
Renata A.G. Reis ◽  
Patricia Ferreira ◽  
Milagros Medina ◽  
M. Cristina Nonato
Keyword(s):  
Steady State ◽  
Leishmania Major ◽  
De Novo ◽  
Enzyme Reaction ◽  
Mechanistic Study ◽  
Rate Limiting Step ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Rate Limiting ◽  
State Kinetic

Leishmania major dihydro-orotate dehydrogenase (DHODHLm) oxidizes dihydro-orotate to orotate (ORO) in the de novo pyrimidine biosynthetic pathway. The enzyme reaction mechanism was elucidated by steady- and pre-steady-state kinetics. ORO release was found to be the rate-limiting step in the overall catalysis.

scholarly journals Interpretation of the Kinetics of Consecutive Enzyme-catalysed Reactions: Studies on the Arginase-Ornithine Carbamoyltransferase System

1982 ◽  
Vol 35 (2) ◽  
pp. 137 ◽  
Author(s):  
RD Teasdale ◽  
PD Jeffrey ◽  
PW Kuchel ◽  
LW Nichol
Keyword(s):  
Steady State ◽  
Ornithine Carbamoyltransferase ◽  
Enzyme Substrate ◽  
Steady State Kinetic ◽  
Kinetic Mechanisms ◽  
Heterogeneous Association ◽  
Kinetics Of ◽  
State Kinetic ◽  
Enzymic Assay

A method that permits the use of measurements on the concentration of the intermediate in a coupled enzymic assay in determining the presence or absence of an interaction between the enzymes is presented. The method is shown to be closely analogous to a previously formulated procedure involving the determination of the rate of production of the final product of such a sequence and is shown to be applicable regardless of the complexity of the operative kinetic mechanisms, provided it may be assumed that all enzyme-substrate complexes are in the steady-state. Kinetic results obtained with the arginase--ornithine carbamoyltransferase couple, in which the intermediate ornithine is monitored, are examined in these terms to conclude that no heterogeneous association is operative between the enzymes.

scholarly journals Kinetics of nitrogenase of Klebsiella pneumoniae. Heterotropic interactions between magnesium-adenosine 5′-diphosphate and magnesium-adenosine 5′-triphosphate

1977 ◽  
Vol 165 (2) ◽  
pp. 255-262 ◽  
Author(s):  
R N F Thorneley ◽  
A Cornish-Bowden
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Protein A ◽  
Competitive Inhibitor ◽  
Electron Transfer Reaction ◽  
H2 Evolution ◽  
Fe Protein ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Kinetics Of

The effects of MgADP and MgATP on the kinetics of a pre-steady-state electron-transfer reaction and on the steady-state kinetics of H2 evulution for nitrogenase proteins of K. pneumoniae were studied. MgADP was a competitive inhibitor of MgATP in the MgATP-induced electron transfer from the Fe-protein to the Mo-Fe-protein. A dissociation constant K′i = 20 micron was determined for MgADP. The release of MgADP or a coupled conformation change in the Fe-protein of K.pneumoniae occurred with a rate comparable with that of electron transfer, k approximately 2 × 10(2)S-1. Neither homotropic nor heterotropic interactions involving MgATP and MgADP were observed for this reaction. Steady-state kinetic data for H2 evolution exhibited heterotropic effects between MgADP and MgATP. The data have been fitted to symmetry and sequential-type models involving conformation changes in two identical subunits. The data suggest that the enzyme can bind up to molecules of either MgATP or MgADP, but is unable to bind both nucleotides simultaneously. The control of H2 evolution by the MgATP/MgADP ratio is not at the level of electron transfer between the Fe- and Mo-Fe-proteins.

scholarly journals HIV reverse transcriptase pre-steady-state kinetic analysis of chain terminators and translocation inhibitors reveals interactions between magnesium and nucleotide 3’-OH

2020 ◽  
Author(s):  
Christopher R. Dilmore ◽  
Jeffrey J. DeStefano
Keyword(s):  
Steady State ◽  
Reverse Transcriptase ◽  
Hiv Reverse Transcriptase ◽  
Oh Groups ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Sugar Ring ◽  
Group 2 ◽  
Equilibrium Dissociation ◽  
State Kinetic

AbstractDeoxythymidine triphosphate analogs with various 3’ groups (-OH (dTTP), -H, -N3, -NH2, -F, -O- CH3, and no group (2′,3′-didehydro-2′,3′-dideoxythymidine triphosphate (d4TTP)), and those retaining the 3’-OH but with 4’ additions (4’-C-methyl, 4’-C-ethyl) or sugar ring modifications (D-carba dTTP) were evaluated using pre-steady-state kinetics in low (0.5 mM) and high (6 mM) Mg2+ with HIV reverse transcriptase (RT). All analogs showed diminished incorporation compared to dTTP ranging from about 2-fold (3’-H, -N3, and d4TTP with 6 mM Mg2+) to >10-fold (3’-NH2 and 3’-F with 0.5 mM Mg2+). The exception was 3’-O-CH3 dTTP which was incorporate profoundly more slowly than other analogs. The incorporation rate (k) using 5 µM dTTP and 0.5 mM (free) Mg2+ was modestly slower (1.6-fold) than with 6 mM Mg2+, while analogs with 3’ modifications were incorporated more slowly (2.8-5.1-fold) in 0.5 mM Mg2+. In contrast, 4’-C-methyl and D-carb, which retain the 3’-OH, were not significantly affected by Mg2+. Consistent with the above results, analogs with 3’ modifications were better inhibitors with 6 mM vs. 0.5 mM Mg2+, in primer extension reactions on a long template. Equilibrium dissociation constant (Kd) and kpol determinations for dTTP and analogs lacking a 3’-OH indicated that low Mg2+ caused a several-fold greater reduction in kpol with the analogs but had little effect on Kd. Overall, results emphasize the importance of as yet undefined interactions between Mg2+ and the 3’-OH and indicate that inhibitors with 3’-OH groups may have an advantage in a physiological setting where the concentration of free Mg2+ is low.

scholarly journals Kinetics of the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis

1984 ◽  
Vol 218 (3) ◽  
pp. 829-833 ◽  
Author(s):  
A Baici ◽  
U Seemüller
Keyword(s):  
Steady State ◽  
Complex Formation ◽  
Rate Constants ◽  
Biological Significance ◽  
Hirudo Medicinalis ◽  
Inhibition Mechanism ◽  
Steady State Kinetic ◽  
Leucocyte Elastase ◽  
Kinetics Of ◽  
State Kinetic

The rate constants for the inhibition of human leucocyte elastase by eglin from the leech Hirudo medicinalis were determined by using a pre-steady-state kinetic approach. kon and koff for complex-formation and dissociation were 1 × 10(6)M-1 X S-1 and 8 × 10(-4)S-1 respectively. Ki was calculated as the ratio koff/kon = 8 × 10(-10)M, the binding of eglin to elastase was reversible and the inhibition mechanism was of the fully competitive type. The mechanistic properties of the system and the biological significance of the rate constants are discussed.

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