Ascorbate Oxidase
René Lontie in Copper Proteins and Copper Enzymes, 1984
The Km values for oxygen and ascorbate and V are constant in the pH region between 4.5 and 7.4.19,62,63 Hexacyanoferrate(II) was also oxidized at a good rate.63 At pH 6 the Km was 3 mM and the turnover number was 1,500. At variance with ascorbate the oxidation of hexacyanoferrate(II) was strongly affected by pH. The turnover number at pH 7.4 was three orders of magnitude lower than at pH 4.5. Also, the Km for hexacyanoferrate(II) was slightly affected, being smaller at lower pH. The apparent affinity for oxygen was different when the substrate was ascorbate or hexacyanoferrate(II). At pH 6 in presence of ascorbate the value of Km was 0.2 mM; in presence of hexacyanoferrate(II) only a limiting value <10 μM was calculated from the progress curves. These results do not fit with a simple “ping-pong” mechanism, as proposed for laccase.53
Hydrolytic Enzymes for the Synthesis of Pharmaceuticals
Peter Grunwald in Pharmaceutical Biocatalysis, 2019
Interestingly, kinetic resolution of their racemic forms produces both chiral epoxides and diols, which are valuable synthetic precursors for the synthesis of pharmaceuticals (Choi and Choi, 2005). One example is Propranolol, a commonly employed β-blocker agent whose (S)-enantiomer displays 100-times more activity than its antipode in blocking β-adrenergic receptors. An evolved epoxide hydrolase (EH) from Bacillus megaterium has been reported to be highly efficient in the hydrolytic resolution of α-naphthyl glycidyl ether (20 g of epoxide in a 100 g/L concentration reaction, Kong et al., 2015). Using a biphasic system composed by iPr2O and isooctane in combination with a buffer and a surfactant such as Tween-80, the corresponding (S)-epoxide (45% yield, STY of 136 g L−1 d−1) and the (R)-diol (42% yield, STY of 139 g L−1 d−1) were both isolated in enantiopure form (Scheme 9.17). Both optically active compounds served for the immediate synthesis of Propranolol enantiomers. A total turnover number of the enzyme of 70000 was reached, which is much higher than the ones described with other enzymatic and non-enzymatic catalysts. Chemoenzymatic synthesis of Propranolol enantiomers by using an engineered epoxide hydrolase.
Disease Prediction and Drug Development
Arvind Kumar Bansal, Javed Iqbal Khan, S. Kaisar Alam in Introduction to Computational Health Informatics, 2019
The reaction rate depends upon the enzyme binding to the substrate. Using partial differential equations, it can be expressed as being proportional to the product of the reactants. The reaction-rate and various coefficients are shown in Equation 10.3. In the reaction, E is the enzyme; S is the substrate; P is the product; κfor is the forward reaction-coefficient; κrev is the reverse reaction-coefficient and κcat is the turnover number – the maximum number of substrate molecules converted to product molecule per second.
In the quest for new targets for pathogen eradication: the adenylosuccinate synthetase from the bacterium Helicobacter pylori
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2018
Ante Bubić, Natalia Mrnjavac, Igor Stuparević, Marta Łyczek, Beata Wielgus-Kutrowska, Agnieszka Bzowska, Marija Luić, Ivana Leščić Ašler
Kinetic constants determined for all three substrates of AdSS, IMP, GTP, and Asp are given in Table 3. For each substrate, the curve of initial velocity over substrate concentration follows Michaelis–Menten kinetics (Equation (1)). When comparing Michaelis constants of H. pylori AdSS with those of other organisms, it can be observed that they are similar to those of e.g. E. coli AdSS (GTP: 10–48 µM, IMP: 20–200 µM, and Asp: 260–350 µM) or mouse basic isozyme (GTP: 12 µM, IMP: 45 µM, and Asp: 140 µM), while e.g. the mouse acidic isozyme (GTP: 15 µM, IMP: 12 µM, and Asp: 950 µM) or P. falciparum AdSS (GTP: 18 µM, IMP: 23 µM, and Asp: 1800 µM) have significantly higher values of Km for Asp1,39. Turnover number (kcat) of 1 s−1 compares well with kcat for E. coli and P. falciparum AdSS enzymes, however, both mouse enzymes have 4- to 5-fold higher efficacy39. This low value of kcat makes AdSS one of the least efficient enzymes described in the literature, and for E. coli the AdSS reaction is near the rate-limiting step in the bacterium’s growth and reproduction cycle3. This is in line with the known fact that purine metabolism is the growth-limiting step for all cells, both prokaryotic and eukaryotic24.
Bile acid oxidation by Eggerthella lenta strains C592 and DSM 2243T
Published in Gut Microbes, 2018
Spencer C. Harris, Saravanan Devendran, Celia Méndez- García, Sean M. Mythen, Chris L. Wright, Christopher J. Fields, Alvaro G. Hernandez, Isaac Cann, Phillip B. Hylemon, Jason M. Ridlon
Previous work characterizing dozens of E. lenta strains, most of which were not deposited in culture collections, demonstrated 12α-HSDH activity.17,19,50 Here, we present a novel gene encoding 12α-HSDH in E. lenta (Figure 4). Purified rElen_2515 converted 12-oxoLCA to a product co-migrating with DCA, whose major mass ion was identical to authentic DCA (Figure 4). Kinetic analysis of the enzyme clearly shows that the oxidative direction is favored with a Km in the oxidative direction ∼100 μM lower than the reductive direction, and a turnover number (Kcat) an order of magnitude higher in the oxidative direction (Table 1). This is consistent with the observed complete oxidation of hydroxyl groups of CA, CDCA and DCA under a nitrogen atmosphere by E. lenta whole cells (Figures 1 & 2). rElen_2515 was NAD(H)-dependent (Table 2) with broad specificity, recognizing unconjugated primary (CA) and secondary bile acids (DCA), as well as taurine-conjugated (TCA, TDCA) and glycine-conjugated bile acids (GDCA, GCA) possessing a 12α-hydroxyl group. CDCA, which lacks a 12α-hydroxyl group was not a substrate (Table 2). This is in contrast to the only other gene reported that encodes 12α-HSDH from Clostridium group P strain C48-50/ATCC 29733, which had greatest specificity for CA and required NADP(H) as co-enzyme.32
Evaluation of the paraoxonase-1 kinetic parameters of the lactonase activity by nonlinear fit of progress curves
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Although basic biochemical and physiological principles dictate that it is the activity of a given enzyme that is important with respect to its function, two kinetic parameters determine the activity of enzyme at given substrate concentration; i.e. limiting rate Vmax (=kcat·[E]T) which depends on turnover number and enzyme active sites concentration, and the Michaelis constant Km which is not concentration dependent characteristics of an enzyme. However, the values of Km are associated with polymorfic forms2, enyzme modifications and its environment.
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