scholarly journals Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase

1985 ◽  
Vol 225 (1) ◽  
pp. 159-165 ◽  
Author(s):  
F M Dickinson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Dissociation Reaction ◽  
Quenching Effect ◽  
Sheep Liver ◽  
Association Reaction ◽  
Hydrolysis Of

The dissociation of the aldehyde dehydrogenase X NADH complex was studied by displacement with NAD+. The association reaction of enzyme and NADH was also studied. These processes are biphasic, as shown by McGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462], but the details of the dissociation reaction are significantly different from those given by those authors. Spectral and kinetic experiments provide evidence for the formation of abortive complexes of the type enzyme X NADH X aldehyde. Kinetic studies at different wavelengths with transcinnamaldehyde as substrate provide evidence for the formation of an enzyme X NADH X cinnamoyl complex. Hydrolysis of the thioester relieves a severe quenching effect on the fluorescence of enzyme-bound NADH.

scholarly journals Steady-state and pre-steady kinetic studies on mitochondrial sheep liver aldehyde dehydrogenase. A comparison with the cytoplasmic enzyme

1978 ◽  
Vol 171 (3) ◽  
pp. 527-531 ◽  
Author(s):  
A K H MacGibbon ◽  
L F Blackwell ◽  
P D Buckley
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Kinetic Properties ◽  
Protein Fluorescence ◽  
Sheep Liver ◽  
Steady State Kinetic ◽  
Wide Range ◽  
Liver Aldehyde Dehydrogenase

Kinetic studies were carried out on mitochondrial aldehyde dehydrogenase (EC 1.2.1.3) isolated from sheep liver. Steady-state studies over a wide range of acetaldehyde concentrations gave a non-linear double-reciprocal plot. The dissociation of NADH from the enzyme was a biphasic process with decay constants 0.6s-1 and 0.09s-1. Pre-steady-state kinetic data with propionaldehyde as substrate could be fitted by using the same burst rate constant (12 +/- 3s-1) over a wide range of propionaldehyde concentrations. The quenching of protein fluorescence on the binding of NAD+ to the enzyme was used to estimate apparent rate constants for binding (2 × 10(4) litre.mol-1.s-1) and dissociation (4s-1). The kinetic properties of the mitochondrial enzyme, compared with those reported for the cytoplasmic aldehyde dehydrogenase from sheep liver, show significant differences, which may be important in the oxidation of aldehydes in vivo.

scholarly journals Kinetic studies on the esterase activity of cytoplasmic sheep liver aldehyde dehydrogenase

1978 ◽  
Vol 171 (3) ◽  
pp. 533-538 ◽  
Author(s):  
A K H MacGibbon ◽  
S J Haylock ◽  
P D Buckley ◽  
L F Blackwell
Keyword(s):  
Steady State ◽  
Aldehyde Dehydrogenase ◽  
Kinetic Studies ◽  
Sheep Liver ◽  
Rate Limiting Step ◽  
Dehydrogenase Reaction ◽  
Steady State Rate ◽  
Michaelis Constants ◽  
State Rate ◽  
Liver Aldehyde Dehydrogenase

The hydrolysis of 4-nitrophenyl acetate catalysed by cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by steady-state and transient kinetic techniques. NAD+ and NADH stimulated the steady-state rate of ester hydrolysis at concentrations expected on the basis of their Michaelis constants from the dehydrogenase reaction. At higher concentrations of the coenzymes, both NAD+ and NADH inhibited the reaction competitively with respect to 4-nitrophenyl acetate, with inhibition constants of 104 and 197 micron respectively. Propionaldehyde and chloral hydrate are competitive inhibitors of the esterase reaction. A burst in the production of 4-nitrophenoxide ion was observed, with a rate constant of 12 +/- 2s-1 and a burst amplitude that was 30% of that expected on the basis of the known NADH-binding site concentration. The rate-limiting step for the esterase reaction occurs after the formation of 4-nitrophenoxide ion. Arguments are presented for the existence of distinct ester- and aldehyde-binding sites.

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 Pre-steady-state kinetic studies on cytoplasmic sheep liver aldehyde dehydrogenase

1977 ◽  
Vol 167 (2) ◽  
pp. 469-477 ◽  
Author(s):  
Alastair K. H. MacGibbon ◽  
Leonard F. Blackwell ◽  
Paul D. Buckley
Keyword(s):  
Steady State ◽  
Isotope Effect ◽  
Aldehyde Dehydrogenase ◽  
Rate Constant ◽  
Kinetic Studies ◽  
Similar Data ◽  
Protein Fluorescence ◽  
Sheep Liver ◽  
Steady State Kinetic ◽  
Liver Aldehyde Dehydrogenase

Stopped-flow experiments in which sheep liver cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) was rapidly mixed with NAD+ and aldehyde showed a burst of NADH formation, followed by a slower steady-state turnover. The kinetic data obtained when the relative concentrations and orders of mixing of NAD+ and propionaldehyde with the enzyme were varied were fitted to the following mechanism: [Formula: see text] where the release of NADH is slow. By monitoring the quenching of protein fluorescence on the binding of NAD+, estimates of 2×105 litre·mol−1·s−1 and 2s−1 were obtained for k+1 and k−1 respectively. Although k+3 could be determined from the dependence of the burst rate constant on the concentration of propionaldehyde to be 11s−1, k+2 and k−2 could not be determined uniquely, but could be related by the equation: (k−2+k+3)/k+2 =50×10−6mol·litre−1. No significant isotope effect was observed when [1-2H]propionaldehyde was used as substrate. The burst rate constant was pH-dependent, with the greatest rate constants occurring at high pH. Similar data were obtained by using acetaldehyde, where for this substrate (k−2+k+3)/k+2=2.3×10 −3mol·litre−1 and k+3 is 23s−1. When [1,2,2,2-2H]acetaldehyde was used, no isotope effect was observed on k+3, but there was a significant effect on k+2 and k−2. A burst of NADH production has also been observed with furfuraldehyde, trans-4-(NN-dimethylamino)cinnamaldehyde, formaldehyde, benzaldehyde, 4-(imidazol-2-ylazo)benzaldehyde, p-methoxybenzaldehyde and p-methylbenzaldehyde as substrates, but not with p-nitrobenzaldehyde.

Micellar catalysis of organic reactions. VIII. Kinetic studies of the hydrolysis of 2-acetyloxybenzoic acid (aspirin) in the presence of micelles

1982 ◽  
Vol 35 (7) ◽  
pp. 1357 ◽  
Author(s):  
TJ Broxton
Keyword(s):  
Aqueous Solution ◽  
Cetyltrimethylammonium Bromide ◽  
Cetylpyridinium Chloride ◽  
Kinetic Studies ◽  
Base Catalysis ◽  
Plateau Region ◽  
General Base ◽  
Mechanism Of Hydrolysis ◽  
Ph Range ◽  
Hydrolysis Of

The hydrolysis of 2-acetyloxybenzoic acid in the pH range 6-12 has been studied in the presence of micelles of cetyltrimethylammonium bromide (ctab) and cetylpyridinium chloride (cpc). In the plateau region (pH 6-8) the hydrolysis is inhibited by the presence of micelles, while in the region where the normal BAC2 hydrolysis (pH > 9) occurs the reaction is catalysed by micelles of ctab and cpc. The mechanism of hydrolysis in the plateau region is shown to involve general base catalysis by the adjacent ionized carboxy group both in the presence and absence of micelles. This reaction is inhibited in the presence of micelles because the substrate molecules are solubilized into the micelle and water is less available in this environment than in normal aqueous solution.

scholarly journals Reaction between sheep liver mitochondrial aldehyde dehydrogenase and various thiol-modifying reagents

1989 ◽  
Vol 261 (1) ◽  
pp. 281-284 ◽  
Author(s):  
K M Loomes ◽  
T M Kitson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Related Compound ◽  
Physiological Responses ◽  
Enzymic Activity ◽  
Cysteine Residues ◽  
Mitochondrial Enzyme ◽  
Step Process ◽  
Cytoplasmic Enzyme ◽  
Sheep Liver

Sheep liver mitochondrial aldehyde dehydrogenase reacts with 2,2′-dithiodipyridine and 4,4′-dithiodipyridine in a two-step process: an initial rapid labelling reaction is followed by slow displacement of the thiopyridone moiety. With the 4,4′-isomer the first step results in an activated form of the enzyme, which then loses activity simultaneously with loss of the label (as has been shown to occur with the cytoplasmic enzyme). With 2,2′-dithiodipyridine, however, neither of the two steps of the reaction has any effect on the enzymic activity, showing that the mitochondrial enzyme possesses two cysteine residues that must be more accessible or reactive (to this reagent at least) than the postulated catalytically essential residue. The symmetrical reagent 5,5′-dithiobis-(1-methyltetrazole) activates mitochondrial aldehyde dehydrogenase approximately 4-fold, whereas the smaller related compound methyl l-methyltetrazol-5-yl disulphide is a potent inactivator. These results support the involvement of mixed methyl disulphides in causing unpleasant physiological responses to ethanol after the ingestion of certain antibiotics.

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