Steady-state and pre-steady-state kinetics of coenzyme A linked aldehyde dehydrogenase from Escherichia coli.
Coenzyme A linked aldehyde dehydrogenase from Escherichia coli strain B has been purified to a specific activity of 14 units/mg of protein, and initial rate and substrate analogue inhibition experiments have been performed. On the basis of these steady-state measurements, a bi-uni-uni-uni ping-pong mechanism is proposed in which NAD+ binds to the free enzyme followed by acetaldehyde. The product NADH is then released before coenzyme A (CoA) can bind, and acetyl-CoA is the final product released. A pre-steady-state time-dependent activation of the enzyme was observed when assays were initiated with NAD+. This lag phase of the reaction was studied as a function of the NAD+ concentration and found to be first order. Furthermore, the presence of NAD+ was demonstrated to be necessary to maintain the enzyme in the active conformation. Evidence that the enzyme contains two distinct NAD+ binding sites, an activator site and a catalytic site, has been obtained from pre-steady-state experiments with the NAD+ analogues AMP and 3-pyridine-carboxaldehyde adenine dinucleotide. AMP, a potent competitive inhibitor with respect to NAD+ under steady-state conditions, did not affect the rate of enzyme activation during pre-steady-state measurements. The analogue 3-pyridine-carboxaldehyde adenine dinucleotide, a potent activator of the aldehyde dehydrogenase, was a poor substrate compared with NAD+. At concentrations of this analogue that fully activated the enzyme, no alternate substrate inhibition was observed with respect to NAD+. A model incorporating two binding sites for NAD+ has been put forward to explain these observations.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000444:Aldehyde Dehydrogenase; D000445:Aldehyde Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000447:Aldehydes / Q000494:pharmacology; D003065:Coenzyme A; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology
D000447:Aldehydes; D011725:Pyridines; D009243:NAD; D000249:Adenosine Monophosphate; C014785:3-pyridinaldehyde; D000445:Aldehyde Oxidoreductases; D000444:Aldehyde Dehydrogenase; D003065:Coenzyme A