Steady-state and pre-steady-state kinetics of propionaldehyde oxidation by sheep liver cytosolic aldehyde dehydrogenase at pH 5.2. Evidence that the release of NADH remains rate-limiting in the enzyme mechanism at acid pH values.
The kcat value for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase increased 3-fold, from 0.16 s-1 at pH 7.6 to 0.49 s-1 at pH 5.2, in parallel with the increase in the rate of displacement of NADH from binary enzyme.NADH complexes. A burst in nucleotide fluorescence was observed at all pH values consistent with the rate of isomerization of binary enzyme.NADH complexes constituting the rate-limiting step in the steady state. No substrate activation by propionaldehyde was observed at pH 5.2, but the enzyme exhibited dissociation/association behavior. The inactive dissociated form of the enzyme was favored by low enzyme concentration, low pH, and low ionic strength. Propionaldehyde protected the enzyme against dissociation.
Hill
J P
JP
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Blackwell
L F
LF
Buckley
P D
PD
Motion
R L
RL
D000444:Aldehyde Dehydrogenase / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000378:metabolism; D009994:Osmolar Concentration; D012756:Sheep; D013050:Spectrometry, Fluorescence
D000447:Aldehydes; D009243:NAD; C005556:propionaldehyde; D000444:Aldehyde Dehydrogenase