Page 2 - U. Protein structure and function

[U. Protein structure and function-1]

The activation of glycerol dehydrogenase by ppGpp

Huyen Nga Hoang¹, Che-Hun Jung¹˙*

¹Molecular Medicine, Chonnam National University, Gwangju 61186, Korea

Glycerol dehydrogenase (GldA) from Escherichia coli is a Zn2+-containing alcohol dehydrogenase, catalyzing the
NAD+-dependent oxidation of glycerol to dihydroxyacetone. Accumulation of ppGpp initiates stringent response of
bacteria under nutritional stress. In this study, ppGpp binds to GldA and activates its activity with the half-maximal

activation at 33.1 ± 3.1 µM. GTP and GDP also bind to GldA with similar affinity to ppGpp, however, they have no

effect on GldA activity considering the physiological conditions. ppGpp activated GldA while
Tris(hydroxymethyl)aminomethane serves as a competitive inhibitor against glycerol with the inhibition constants
(Ki) of 410 ± 21 µM. The interactions of E. coli GldA with its substrates and products were examined by both intrinsic

fluorescence quenching and enzyme kinetics studies. The dissociation constants of NAD+, NADH, and
dihydroxyacetone for GldA were 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. Our results suggest that the intrinsic fluorescence
change of proteins by ligand binding is sufficiently sensitive for studying protein-ligand interactions.