Competition of two substrates for a single enzyme. A simple kinetic theorem exemplified by a hydroxy steroid dehydrogenase reaction

Pocklington, T; Jeffery, Jonathan

doi:10.1042/bj1120331

Cited by 38 publications

(22 citation statements)

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“…Accordingly, PP1 exchange was studied in a competition experiment with various concentrations of ATP and dATP (Fig. 4); the kinetics of this experiment were similar to those described by Pocklington & Jeffery (1969) Purified ATP sulphurylase did not catalyse PPi exchange when sulphate was replaced with 1 mM-Lcysteine, L-methionine, glycine or a mixture of 20 protein amino acids (L-isomers, each 1 mM), indicating that the enzyme was separated from spinach-leaf aminoacyl-tRNA synthetases (Marcus, 1959). The enzyme was also separated from the short-chain fatty acid thiokinases of spinach-leaf tissue (Millerd & Bonner, 1953) …”

Section: Properties Ofpurified Atp Sulphurylasementioning

confidence: 97%

Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

Shaw

Anderson

1972

Biochemical Journal

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Section: Properties Ofpurified Atp Sulphurylasementioning

confidence: 97%

Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

Shaw

Anderson

1972

Biochemical Journal

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“…Our final assumption includes competitive inhibition of bile acids for both the uptake and efflux transporters using the adapted formula for enzymes (Asante-Appiah and Chan, 1996;Martinez-Irujo et al, 1998;Pocklington and Jeffery, 1969;Schäuble et al, 2013). In addition, the drugs we choose as model drugs, glibenclamide and cyclosporin A, are both competitive inhibitors of BSEP.…”

Section: Development Of the Mechanistic Biokinetic Modelmentioning

confidence: 99%

Development of a mechanistic biokinetic model for hepatic bile acid handling to predict possible cholestatic effects of drugs

Notenboom

Weigand

Proost

et al. 2018

European Journal of Pharmaceutical Sciences

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A B S T R A C TDrug-induced liver injury (DILI) is a common reason for drug withdrawal from the market. An important cause of DILI is drug-induced cholestasis. One of the major players involved in drug-induced cholestasis is the bile salt efflux pump (BSEP; ABCB11). Inhibition of BSEP by drugs potentially leads to cholestasis due to increased (toxic) intrahepatic concentrations of bile acids with subsequent cell injury. In order to investigate the possibilities for in silico prediction of cholestatic effects of drugs, we developed a mechanistic biokinetic model for human liver bile acid handling populated with human in vitro data. For this purpose we considered nine groups of bile acids in the human bile acid pool, i.e. chenodeoxycholic acid, deoxycholic acid, the remaining unconjugated bile acids and the glycine and taurine conjugates of each of the three groups. Michaelis-Menten kinetics of the human uptake transporter Na + -taurocholate cotransporting polypeptide (NTCP; SLC10A1) and BSEP were measured using NTCP-transduced HEK293 cells and membrane vesicles from BSEP-overexpressing HEK293 cells. For in vitro-in vivo scaling, transporter abundance was determined by LC-MS/MS in these HEK293 cells and vesicles as well as in human liver tissue. Other relevant human kinetic parameters were collected from literature, such as portal bile acid levels and composition, bile acid synthesis and amidation rate. Additional empirical scaling was applied by increasing the excretion rate with a factor 2.4 to reach near physiological steady-state intracellular bile acid concentrations (80 μM) after exposure to portal vein bile acid levels. Simulations showed that intracellular bile acid concentrations increase 1.7 fold in the presence of the BSEP inhibitors and cholestatic drugs cyclosporin A or glibenclamide, at intrahepatic concentrations of 6.6 and 20 μM, respectively. This simplified model provides a tool for a first indication whether drugs at therapeutic concentrations might cause cholestasis by inhibiting BSEP.

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“…The concentration of Pf-AlaRS was adjusted to account for 25% of the final rate of PPi synthesis in the presence of the four non-cognate aa used in the final reaction mixture. Predetermination of the K M and k cat values for each substrate of a given enzyme can be used to predict the effect of individual components on the overall rate of the reaction (i.e., using equations describing the kinetics of the enzymes in the presence of two competing substrates) [38]. …”

Section: Resultsmentioning

confidence: 99%

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

Grube

Roy

2017

RNA Biology

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Aminoacyl-tRNA synthetases (aaRSs) catalyze the aminoacylation of tRNAs to produce the aminoacyl-tRNAs (aa-tRNAs) required by ribosomes for translation of the genetic message into proteins. To ensure the accuracy of tRNA aminoacylation, and consequently the fidelity of protein synthesis, some aaRSs exhibit a proofreading (editing) site, distinct from the aa-tRNA synthetic site. The aaRS editing site hydrolyzes misacylated products formed when a non-cognate amino acid is used during tRNA charging. Because aaRSs play a central role in protein biosynthesis and cellular life, these proteins represent longstanding targets for therapeutic drug development to combat infectious diseases. Most existing aaRS inhibitors target the synthetic site, and it is only recently that drugs targeting the proofreading site have been considered. In the present study, we developed a robust assay for the high-throughput screening of libraries of inhibitors targeting both the synthetic and the proofreading sites of up to four aaRSs simultaneously. Thus, this assay allows for screening of eight distinct enzyme active sites in a single experiment. aaRSs from several prominent human pathogens (i.e., Mycobacterium tuberculosis, Plasmodium falciparum, and Escherichia coli) were used for development of this assay.

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Competition of two substrates for a single enzyme. A simple kinetic theorem exemplified by a hydroxy steroid dehydrogenase reaction

Cited by 38 publications

References 6 publications

Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue

Development of a mechanistic biokinetic model for hepatic bile acid handling to predict possible cholestatic effects of drugs

A continuous assay for monitoring the synthetic and proofreading activities of multiple aminoacyl-tRNA synthetases for high-throughput drug discovery

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