Purification, properties, and kinetic mechanism of coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri

Abstract

Coenzyme A-linked aldehyde dehydrogenase from Clostridium kluyveri was purified from the soluble fraction of crude extracts and its physical and kinetic properties were studied. The enzyme was purified approximately 90-fold over crude extracts to a specific activity of 50 units/mg protein and was estimated to be 40% pure by polyacrylamide gel electrophoresis. From active enzyme centrifugation studies, aldehyde dehydrogenase was found to have a sedimentation coefficient of s 20, w = 7.4. The Stokes radius of the enzyme was determined by gel filtration and found to be 9.5 nm in the presence of substrates and 11.0 nm in the absence of substrates. Using the values found for the sedimentation coefficient and the Stokes radius, the molecular weight of the enzyme in the presence of substrates was calculated to be 290,000 and the frictional ratio, 2.2. Aldehyde dehydrogenase can utilize thiols other than CoA as acetyl acceptors. A number of methods were employed in order to exclude the possibility that these thiols act merely by recycling nonenzymatically trace amounts of CoA that might be in the enzyme preparation. From steady-state kinetic measurements, a ping pong mechanism was proposed in which NAD + binds to free enzyme, acetaldehyde binds next, and NADH is released before CoA binds and acetyl-CoA released. At K m levels of other substrates, substrate inhibition by CoA was observed. The nature of the substrate inhibition is discussed.