Conformational changes and activation of yeast aldehyde dehydrogenase by various agents.
Activity assays are characterised by a slow activation phase lasting 1-2 min which arises from the conversion of the low activity/inactive form of the isolated yeast aldehyde dehydrogenase into a high activity form upon binding NADP(+). When Mg(2+) is present as an activator, the binding of coenzyme is relatively strong and activation is effectively complete when the steady-state rates are measured. Lineweaver-Burk plots with 1/[NADP(+)] as variable are linear. In the absence of Mg(2+), coenzyme binding is relatively weak and the conversion of inactive to active enzyme is not complete when steady-state rates are measured. The balance of equilibrium is now finely balanced and the degree of activation depends on the NADP(+) concentration. Under these conditions, rate versus [NADP(+)] plots are sigmoid, characterised by a Hill coefficient of 1.8 because the active (as opposed to the total) enzyme concentration is different in assays with different [NADP(+)]. Kinetic studies indicate that the activation process involves two steps, but the data do not allow discrimination between alternative mechanisms. Gel filtration and sedimentation equilibrium centrifugation indicate that the activation process does not involve enzyme association-dissociation. Activation of the enzyme can also be achieved by replacing Mg(2+) by small amounts of protamine sulphate, poly-L-lysine or poly-L-arginine. The mechanism for these activations is unknown, but presumably involves the binding of the enzyme to these positively charged molecules.
Dickinson
F Mark
FM
Department of Biological Sciences, University of Hull, Cottingham Road, HU67RX, Hull, UK. f.m.dickenson@hull.ac.uk
D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D004789:Enzyme Activation; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D009249:NADP; D000444:Aldehyde Dehydrogenase