A comparison of nitrophenyl esters and lactones as substrates of cytosolic aldehyde dehydrogenase

Abstract

1. p-Nitrophenyl (PNP) acetate and propionate show a burst of p-nitrophenoxide release when their hydrolysis is catalysed by sheep liver cytosolic aldehyde dehydrogenase. This is not seen in the presence of NAD+ or NADH, implying a change in ratedetermining step. 2. 6-Nitrodihydrocoumarin (6-NDC) shows no burst of absorbance in the visible region. We propose that the pKa of the transient "reporter group' produced during the hydrolysis of this lactone is high (approx. 10) and that the incipient covalently linked p-nitrophenoxide moiety is protonated immediately on formation. The small burst seen in the hydrolysis of 5-nitro-2-coumaranone (5-NC) suggests that the pKa of its reporter group is about 8.5. 3. NADH markedly enhances the steady-state rate with the lactones. 5-NC shows a large rapid burst of colour development in the presence of NADH; this implies that NADH decreases the pKa of the reporter group to 7-7.5. 4. In the presence of NAD+, 5-NC and 6-NDC give an unusual "negative burst' in the stopped-flow traces. We propose that, under these circumstances, acylation of the enzyme is extremely fast and that the first event seen in the stopped-flow traces is protonation of the reporter group. NAD+ also greatly increases the steady-state rate. 5. With the lactones in the presence of NADH, the kcat value (nearly 6 s-1), a measure of the deacylation rate, is compatible with the single-site model for dehydrogenase and esterase activities.

The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases

“…Chromogenic substrates have been used in screening protocols (21), to study substrate specificities of esterases (22), phosphatases (23), dehydrogenases (24), and peptidases (25). The method described in this paper should also be applicable to obtain kinetic parameters for the conversion of invisible substrates by those enzymes.…”

Covalent Modification Reactions Involving Cytosolic Aldehyde Dehydrogenase and a Variety of Resorufin Derivatives

“…Cytosolic aldehyde dehydrogenase was isolated from sheep liver as previously described (4). Resorufin acetate was prepared as before (10); resorufin dimethylcarbamate, resorufin methanesulphonate, and resorufin ethyl ether were prepared as described in the accompanying paper (11).…”

Studies of the esterase activity of cytosolic aldehyde dehydrogenase with resorufin acetate as substrate

“…Only at very high concentrations of acetaldehyde is the rate reduced ; the rate of hydrolysis of resorufin acetate (25 µM) in the presence of NAD + (0.1 mM) and the following concentrations of acetaldehyde is given as a percentage of the control rate in the absence of acetaldehyde : 1 mM (104 %), 10 mM (79 %), 20 mM (52 %), 50 mM (21 %). For a variety of reasons we think that the esterase and dehydrogenase activities of aldehyde dehydrogenase occur at the same active site [4,15,16] ; we must therefore provide an explanation of why acetaldehyde at a concentration (1 mM) that would be considered fully saturating is ineffective as an inhibitor of the esterase activity. During the course of the aldehyde dehydrogenation reaction catalysed by the enzyme, the slow step is the dissociation of NADH from the E:NADH complex.…”

“…In previous work [16] we explained the effect of NAD + on the hydrolysis of PNP acetate by aldehyde dehydrogenase in terms of a change in rate-determining step. Low concentrations of NAD + enhance the initially rate-limiting deacylation step (i.e.…”