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Enzyme Kinetics and Drugs as Enzyme Inhibitors
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Competitive inhibitors are of widespread clinical use as therapeutics. In case of competitive inhibition, the active site of the enzyme binds either the substrate (ES) or the inhibitor (EI) but not both inhibitor and substrate (ESI). Competitive inhibitors show structural similarities with the natural substrate and are recognized by the active site. Competitive inhibition can be reversed by increasing the substrate concentration. Product inhibition can be looked at as a special kind of competitive inhibition; for example, hexokinase binds its substrate glucose as well as the product glucose-6-phosphate so that an accumulation of this metabolite is avoided. The rate equation for competitive inhibition is
Pharmacodynamics of Anticoagulants
Published in Hartmut Derendorf, Günther Hochhaus, Handbook of Pharmacokinetic/Pharmacodynamic Correlation, 2019
Dennis Mungall, Richard H. White
Deswart and coworkers166 have described heparin kinetics utilizing a model combining a saturable and first-order elimination. They were able to fit both single-dose and continuous infusion data for heparin. They also suggested the possibility of utilizing a product inhibition model. To date no metabolite of heparin has been identified that will inhibit the metabolism of heparin. Mungall et al.188 compared a linear kinetic model with a Michaelis-Menten model in 44 patients with proximal vein thrombosis. The Michaelis-Menten model was superior to the linear model. The Vmax for men was 3555 ± 2139 units/h and for women 2373 ± 2000 units/h. The Km was similar for men (0.34 units/ml) and women (.35 units/ml).
Framework
Published in Peter W. Hochachka, Muscles as Molecular and Metabolic Machines, 2019
With regard to regulation of catalytic function, most of the major catalytic differences between M4 and H4 isozymes are attributed to several substitutions in the coenzyme binding domain. These substitutions are considered necessary to account for (i) altered affinities for pyruvate and NADH, (ii) altered turnover number, and (iii) altered sensitivity to product inhibition. Altered histidine content and altered net charges of M4 and H4 LDHs (i.e., altered buffering capacity and possibly altered intracellular binding locations) are determined by changes in more than one domain of each subunit (Baldwin, 1988). Hence, if the LDHs are typical (i.e., are indeed an archetypal isozyme system), then in terms of amounts and kinds of adaptations occurring during evolution of fiber specific and isozyme specific glycolytic pathways, it is fair to conclude that many substitutions must be incorporated at several steps in the pathway from genes to unique protein isoforms to account for the transition from slow to fast kinds of muscle fibers.
Mixed and non-competitive enzyme inhibition: underlying mechanisms and mechanistic irrelevance of the formal two-site model
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Resorting to authoritative sources certainly reduces the risk of misapprehension, but for those who do not have a strong background in enzymology, attaining a proper understanding of mixed inhibition and its molecular determinants remains arduous. For example, Cornish-Bowden in “Fundamental of Enzyme Kinetics” clearly explains the limits of the formal two-site mechanism and suggests that mixed inhibition occurs mainly as a case of product inhibition with iso-mechanism enzymes12. Johnson, in “Kinetic Analysis for the New Enzymology”, adds that mixed inhibition, analogous to uncompetitive inhibition, occurs mainly with multi-substrate reactions, either as a form of product inhibition or, for ternary-complex mechanisms, when a dead-end inhibitor binds before the variable substrate.7 Copeland instead, in “Evaluation of Enzyme Inhibition in Drug Discovery”, mentions five mechanisms through which active site-directed inhibitors can cause inhibition patterns typical of mixed inhibition.3 However, these five mechanisms are treated as exceptions to a general rule. With the general rule being, again, the formal two-site mechanism.
Prediction of long-term polysorbate degradation according to short-term degradation kinetics
Published in mAbs, 2023
Sisi Zhang, Caterina Riccardi, Dane Carlson, Douglas Kamen, Kenneth S. Graham, Mohammed Shameem, Hanne Bak, Hui Xiao, Ning Li
We assumed that the KM of PS degradation by LAL was relatively small with respect to the initial substrate concentration, which usually ranges between 0.05% and 0.2% PS in DPs. Therefore, at the beginning of the incubation, the reaction rate (v) was considered to be constant and independent of the substrate concentration [S], and the increased FFA concentration plotted against incubation time was linear. Linearity of the plot began to be lost only when the percentage of PS remaining had substantially decreased and became comparable to the KM, where v began to be dependent on the substrate concentration and to decrease over time. The possible reasons for the decreased reaction rate included substrate (PS) consumption, product inhibition (FFA accumulation) or decreased enzyme activity. In our case, the reaction plot was no longer linear when the percentage of PS20 remaining decreased to 0.024%, or the percentage of PS80 remaining decreased to 0.078%, at 24 months.
Comparisons between human and rodent hepatic glutathione S-Transferase activities reveal sex and species differences
Published in Xenobiotica, 2023
Michael J. Doerksen, Denny Seo, Alexander D. Smith, Robert S. Jones, Michael W.H Coughtrie, Abby C. Collier
Glutathione S-transferase activity was determined using general and isoform-selective substrates through a microplate-based modification of the method described by González et al. (1989), with Beer-Lambert law and published molar extinction coefficients (ε) by Habig et al. (1974) for rate quantification. Briefly, optically clear microplates were placed on ice and the following materials were added: substrate (1 μL of 50 mM stock), protein (10 μL of 2 mg/mL stock), and 0.1 M potassium phosphate buffer (79 μL). The microplate was incubated for 2 min at 37 °C and the reaction was initiated by the addition of 10 mM glutathione (10 μL) for a final volume of 100 μL. The pH of 0.1 M potassium phosphate buffer was 6.5 for all GST assays except for the GST-M assay where the buffer was pH 7.5, as described by Habig et al. (1974). Cibacron Blue, a known inhibitor of GSTs (Tahir et al. 1985; Alin et al. 1985; Warholm et al. 1986), and incubations without the addition of glutathione (no GSH) were used as negative controls. Enzyme activities were determined within 10% of substrate turnover to measure maximum initial reaction velocities and avoid product inhibition. All samples were assayed in triplicate on at least three separate occasions, and the mean activities were reported.