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.

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.

scholarly journals Chorismate synthase. Pre-steady-state kinetics of phosphate release from 5-enolpyruvylshikimate 3-phosphate

1990 ◽  
Vol 265 (3) ◽  
pp. 899-902 ◽  
Author(s):  
T R Hawkes ◽  
T Lewis ◽  
J R Coggins ◽  
D M Mousdale ◽  
D J Lowe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Steady State ◽  
Lag Phase ◽  
Chorismate Synthase ◽  
Phosphate Release ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting ◽  
Kinetics Of

The pre-steady-state kinetics of phosphate formation from 5-enolpyruvylshikimate 3-phosphate catalysed by Escherichia coli chorismate synthase (EC 4.6.1.4) were studied by a rapid-acid-quench technique at 25 degrees C at pH 7.5. No pre-steady-state ‘burst’ or ‘lag’ phase was observed, showing that phosphate is released concomitant with the rate-limiting step of the enzyme. The implications of this result for the mechanism of action of chorismate synthase are discussed.

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 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 Steady-state kinetic mechanism of bovine brain tubulin: tyrosine ligase

1992 ◽  
Vol 286 (1) ◽  
pp. 243-251 ◽  
Author(s):  
N L Deans ◽  
R D Allison ◽  
D L Purich
Keyword(s):  
Steady State ◽  
Thermal Inactivation ◽  
Enzyme Reaction ◽  
Kinetic Mechanism ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Maximal Velocity ◽  
Steady State Kinetic ◽  
Tubulin Tyrosine Ligase ◽  
State Kinetic

The ATP-dependent resynthesis of tubulin from tyrosine and untyrosinated tubulin was examined to establish the most probable steady-state kinetic mechanism of the tubulin: tyrosine ligase (ADP-forming). Three pair-wise sets of initial rate experiments, involving variation of two substrates pair-wise with the third substrate held at a high (but non-saturating) level, yielded convergent-line data, a behaviour that is diagnostic for sequential mechanisms. Michaelis constants were 14 microM, 1.9 microM and 17 microM for ATP, untyrosinated tubulin and L-tyrosine respectively, and the maximal velocity was 0.2 microM/min. AMP was a competitive inhibitor with respect to ATP, and a non-competitive inhibitor versus either tubulin or tyrosine. Likewise, L-dihydroxyphenylalanine acted competitively relative to tyrosine and non-competitively with respect to either ATP or tubulin. These findings directly support a random sequential mechanism. Product inhibition patterns with ADP were also consistent with this assignment; however, inhibition studies were not practical with either orthophosphate or tyrosinated tubulin because both were very weak inhibitors. Substrate protection of the enzyme against alkylation by N-ethylmaleimide and thermal inactivation, along with evidence of enzyme binding to ATP-Sepharose and tubulin-Sepharose, also supports the idea that this three-substrate enzyme reaction exhibits a random substrate addition pathway.

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.

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