Studies on glycerol dehydrogenase from Aerobacter aerogenes

• Main Author: Turner, William
• Published: University of Bath 1973
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.767478

Part I: The Purification and Properties of Glycerol Dehydrogenase. Glycerol dehydrogenase (E C 1.1.1.6) from Aerobacter aerogenes has been purified to homogeneity as judged by starch gel electrophoresis with 60-70% recovery. The enzyme is activated by preincubation with Mn2+. No other divalent cation was found to activate the enzyme although Mg2+, Ca2+, CO2+ and NAD prevented Mn2+ activation. The monovalent cation requirement for glycerol dehydrogenase was studied. K+ and NH4+ were far more efficient activators than Na+ or Li+ although at pH 9.5, K+, Na+ and Li+ had an optimum concentration range for activation. NH4+ did not inhibit enzyme activity over the concentration range tested. Glycerol dehydrogenase was irreversibly inactivated in buffers of low ionic strength. The enzyme was reversibly inactivated by preincubation at 30°C prior to assay. The apparent Km values for dihydroxyacetone, NAD and NADH were determined for both Mn2+- activated and unactivated enzyme. The kinetics of glycerol with glycerol dehydrogenase exhibited substrate activation with both Mn2+- activated and unactivated enzyme. With unactivated enzyme several discrete phases were observed in both Lineweaver Burk and glycerol versus initial rate plots. Hill plots of these data were either non linear or had coefficients of less than one. The data for Mn2+- activated enzyme yielded Hill coefficients of one. These results are consistent with an explanation of substrate activation based on negative co-operativity.;Part II: A Study of the Factors Controlling the Synthesis of Glycerol Dehydrogenase in Vivo. Glycerol dehydrogenase and dihydroxyacetone kinase were both induced by either glycerol or dihydroxyacetone and were both repressed by aerobic growth conditions. Glycerol dehydrogenase was repressed in cells grown in a fermentor between 10% and 75% O2 saturation. At 100% O2 saturation the enzyme was super-repressed. Inactivation of the enzyme occurred in late log phase cells grown above 25% O2 saturation. A much slower rate of enzyme inactivation was observed in cells incubated on a rotary shaker either in the presence of chloramphenicol or in non-proliferating media. Both 10 -4m azide and carbon monoxide prevented the aerobic repression of glycerol dehydrogenase synthesis. Glycerol dehydrogenase was also repressed by glucose and nitrate (but not by fumarate) under anaerobic growth conditions and 10-3M cyclic AMP prevented the glucose repression. Glucose and nitrate acted synergistically in repressing glycerol dehydrogenase but cyclic AMP had little effect in preventing this synergistic repression. It has been proposed that the repression of glycerol dehydrogenase in the presence of oxygen or nitrate is in some way related to the intracellular ATP : ADP ratio. Glucose repression on the other hand is thought to be related to the intracellular cyclic AMP level. On the basis of these proposals at least two different mechanisms controlling glycerol dehydrogenase synthesis are postulated.