scholarly journals Kinetics of p-nitrophenyl pivalate hydrolysis catalysed by cytoplasmic aldehyde dehydrogenase

1989 ◽  
Vol 257 (2) ◽  
pp. 573-578 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Dehydrogenase Activity ◽  
Aldehyde Dehydrogenase ◽  
Chloral Hydrate ◽  
Enzyme Hydrolysis ◽  
Stopped Flow ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Inhibition Pattern ◽  
Rate Determining Steps

The effects of modifiers (NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol and p-nitrobenzaldehyde) on the hydrolysis of p-nitrophenyl (PNP) pivalate (PNP trimethylacetate) catalysed by cytoplasmic aldehyde dehydrogenase are reported. In each case a different inhibition pattern is obtained to that observed when the substrate is PNP acetate; for example, propionaldehyde and chloral hydrate competitively inhibit the hydrolysis of PNP acetate, but are mixed inhibitors with PNP pivalate. The kinetic results can be rationalized in terms of different rate-determining steps: acylation of the enzyme in the case of the pivalate but acyl-enzyme hydrolysis for the acetate. This is confirmed by stopped-flow studies, in which a burst of p-nitrophenoxide is observed when the substrate is PNP acetate, but not when it is the pivalate. PNP pivalate inhibits the dehydrogenase activity of the enzyme competitively with the aldehyde substrate; this is most simply explained if the esterase and dehydrogenase reactions occur at a common enzymic site.

scholarly journals The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme

1986 ◽  
Vol 233 (3) ◽  
pp. 877-883 ◽  
Author(s):  
F M Dickinson ◽  
G W Haywood
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Enzyme Hydrolysis ◽  
Stopped Flow ◽  
Sheep Liver ◽  
Dehydrogenase Reaction ◽  
Liver Aldehyde Dehydrogenase ◽  
Hydrolysis Of ◽  
Flow Experiments ◽  
Esterase Activities

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.

scholarly journals The action of cytoplasmic aldehyde dehydrogenase on methyl p-nitrophenyl carbonate and p-nitrophenyl dimethylcarbamate

1989 ◽  
Vol 257 (2) ◽  
pp. 579-584 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Chloral Hydrate ◽  
Slow Release ◽  
Cysteine Residue ◽  
Single Type ◽  
Rate Of Hydrolysis ◽  
Rate Limiting ◽  
Hydrolysis Of ◽  
Enzyme Intermediate ◽  
Esterase Activities

Cytoplasmic aldehyde dehydrogenase catalyses the hydrolysis of methyl p-nitrophenyl (PNP) carbonate at an appreciable rate that is markedly stimualted by NAD+ or NADH. The nuleotides accelerate the rate-limiting hydrolysis of the acyl-enzyme intermediate while slowing the observed burst of p-nitrophenoxide production. With PNP dimethylcarbamate the enzyme catalyses the slow release of approx. 1 molecule of p-nitrophenoxide per tetrameric enzyme molecule; during the reaction the enzyme becomes effectively inactivated, as the rate of hydrolysis of the acyl-enzyme is virtually zero. The presence of NAD+, NADH, propionaldehyde, chloral hydrate, diethylstilboestrol or disulfiram slows the reaction of enzyme with PNP dimethylcarbamate. The reaction appears to be dependent on a group of pKa 7.6, possibly a cysteine residue. The effect of PNP dimethylcarbamate on the dehydrogenase activity of the enzyme is consistent with there being a single type of active site for the enzyme's dehydrogenase and esterase activities. Steric and electronic factors that govern reaction of the enzyme with PNP substrates are discussed.

Étude cinétique de la N-chloration de l'acide cyanurique en phase aqueuse

1990 ◽  
Vol 68 (2) ◽  
pp. 307-313 ◽  
Author(s):  
Denise Matte ◽  
Bernard Solastiouk ◽  
André Merlin ◽  
Xavier Deglise
Keyword(s):  
Aqueous Medium ◽  
Rate Constants ◽  
Cyanuric Acid ◽  
Stopped Flow ◽  
Experimental Values ◽  
Kinetics Of Reaction ◽  
Limiting Case ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
Phase Aqueuse

A complete kinetic study of the N-chlorination, in basic aqueous medium, of cyanuric acid by the stopflow spectrophotometric method is presented. Our experimental results can be interpreted by two pairs of kinetically indistinguishable mechanisms.[Formula: see text]or[Formula: see text]and[Formula: see text]or[Formula: see text]The rate constants and their corresponding activation energies were determined in the limiting case where one of the elementary steps in each pair is involved alone in the kinetics of reaction.[Formula: see text]The rate constants for hydrolysis of the monochlorinated cyanuric acid derivatives were derived from our experimental values. Keywords: kinetics, N-chlorination, 1,3,5-triazine-2,4,6-trione, aqueous medium, stopped flow.

THE TRYPSIN CATALYZED HYDROLYSIS OF p-NITROPHENYL ACETATE

1959 ◽  
Vol 37 (4) ◽  
pp. 751-759 ◽  
Author(s):  
James A. Stewart ◽  
Ludovic Ouellet
Keyword(s):  
Active Site ◽  
Initial Rate ◽  
Competitive Inhibition ◽  
Early Stage ◽  
Stopped Flow ◽  
Hydrogen Ions ◽  
Experimental Conditions ◽  
Influence Of Ph ◽  
Kinetics Of ◽  
Hydrolysis Of

The hydrolysis of p-nitrophenyl acetate (NPA) by trypsin has been investigated in the early stage of the reaction using stopped-flow techniques. The influence of pH on the initial rate suggests competitive inhibition of the active site of the enzyme by hydrogen ions. The dissociation constant of the enzyme obtained from the kinetics of this reaction (pK = 6.9) indicates possible catalysis by an ammo group or an imidazole group of the enzyme. Lysine methyl ester as an analogue of the enzyme catalyzes the hydrolysis of NPA under similar experimental conditions. The results are described in terms of an assumed mechanism and the nature of the catalytic site is discussed.

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

1996 ◽  
Vol 316 (1) ◽  
pp. 225-232 ◽  
Author(s):  
Trevor M. KITSON ◽  
Kathryn E. KITSON
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Visible Region ◽  
Stopped Flow ◽  
Site Model ◽  
Rate Determining Step ◽  
Steady State Rate ◽  
State Rate ◽  
Reporter Group ◽  
Hydrolysis Of

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 rate-determining 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.

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