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- W2996702783 abstract "Glycerol dehydrogenase (GldA) from Escherichia coli is a Zn2+-containing alcohol dehydrogenase which catalyzes the NAD+-dependent oxidation of glycerol to dihydroxyacetone. In this study, GldA has been cloned, over-expressed, and isolated by an affinity and an ion-exchange chromatography. GldA shows a strong intrinsic fluorescence at 320 nm, when excited at 280 nm. The fluorescence intensity decreases in the presence of NAD+, NADH, and dihydroxyacetone, the substrate and products for GldA, which allows us to determine the dissociation constants for those molecules as 110.6 ± 5.0 μM, 9,1 ± 0.6 μM, 33.3 ± 2.3 mM, respectively. The dissociation constant for NAD+ was similar to the kinetic constant, KM. Guanosine-5′-diphosphate 3′-diphosphate (ppGpp), accumulated in E. coli when starved for amino acids, nutrients, and phosphate, serves as a global regulator in replication, transcription, and translation. In this study, the fluorescence intensity of GldA also decreases in the presence of ppGpp and the dissociation constant for ppGpp is calculated as 108.9 ± 8.6 μM. ppGpp increases GldA activity with the half maximal activation at 33.1 ± 3.1 μM. On the contrary, GTP and GDP inhibit GldA, with the inhibition constants of 16.1 ± 1.1 mM and 10.6 ± 0.3 mM, respectively. Tris(hydroxymethyl)aminomethane serves as a competitive inhibitor against glycerol. GTP and GDP also bind to GldA with the dissociation constants of 60.0 ± 0.8 and 61.0 ± 1.3 μM, respectively. These results suggest that GTP and GDP bind to GldA as strongly as ppGpp but only ppGpp activates GldA. This study shows that ppGpp binds to GldA and activates its activity for the first time. It is also suggested that the strong intrinsic fluorescence of enzymes and their changes in the presence of various ligands can be utilized to measure the binding affinities for those ligands. The method described here is especially effective for bindings with the relatively lower affinities." @default.
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- W2996702783 date "2019-12-17" @default.
- W2996702783 modified "2023-09-25" @default.
- W2996702783 title "The Activation of Glycerol Dehydrogenase from<scp><i>Escherichia coli</i></scp>by ppGpp" @default.
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- W2996702783 doi "https://doi.org/10.1002/bkcs.11932" @default.
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