Use of a Chromophoric Reporter Group to Probe the Active Site of Cytosolic Aldehyde Dehydrogenase

1995 ◽  
pp. 35-43
Author(s):  
Trevor M. Kitson ◽  
Kathryn E. Kitson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Reporter Group

scholarly journals Probing the active site of cytoplasmic aldehyde dehydrogenase with a chromophoric reporter group

1994 ◽  
Vol 300 (1) ◽  
pp. 25-30 ◽  
Author(s):  
T M Kitson ◽  
K E Kitson
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Negative Charge ◽  
Positive Charge ◽  
Gel Filtration ◽  
Modified Enzyme ◽  
Covalently Linked ◽  
Reporter Group

3,4-Dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one (‘DMNB’) reacts with cytoplasmic aldehyde dehydrogenase in a similar way to that previously observed with the structurally related p-nitrophenyl dimethylcarbamate, but provides a covalently linked p-nitrophenol-containing reporter group at the enzyme's active site. The pKa of the enzyme-linked reporter group is much higher than that of free p-nitrophenol, which is consistent with its being in a very hydrophobic environment, or possibly one containing negative charge. Upon binding of NAD+ to the modified enzyme, the pKa falls dramatically, by about 4 1/2 pH units. This implies that under these conditions there is a positive charge near the p-nitrophenoxide moiety, perhaps that of the nicotinamide ring of NAD+. The modified enzyme binds NAD+ very tightly; neither gel filtration nor dialysis is effective in separating them. However, the reporter group provides a convenient way of monitoring the displacement of this bound NAD+ when NADH is added.

scholarly journals Aldehyde dehydrogenase. An enzyme with two distinct catalytic activities at a single type of active site

1985 ◽  
Vol 230 (1) ◽  
pp. 261-267 ◽  
Author(s):  
R J Duncan
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Active Sites ◽  
Catalytic Site ◽  
Single Type ◽  
Catalytic Activities ◽  
Binding Domains

The evidence for and against the esterase and dehydrogenase active sites of aldehyde dehydrogenase being topologically distinct is examined. It is found that all the evidence (including all that previously amassed by others in favour of distinct binding domains) is actually consistent with, and in favour of, a single type of catalytic site having both activities. The existence of separate high-Km modulating sites for the enzyme is also questioned.

Novel NADPH–cysteine covalent adduct found in the active site of an aldehyde dehydrogenase

2011 ◽  
Vol 439 (3) ◽  
pp. 443-455 ◽  
Author(s):  
Ángel G. Díaz-Sánchez ◽  
Lilian González-Segura ◽  
Enrique Rudiño-Piñera ◽  
Alfonso Lira-Rocha ◽  
Alfredo Torres-Larios ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Cysteine Residue ◽  
Covalent Modification ◽  
Enzyme Inactivation ◽  
Betaine Aldehyde Dehydrogenase ◽  
Betaine Aldehyde ◽  
Oxidative Stresses ◽  
Covalent Adduct

PaBADH (Pseudomonas aeruginosa betaine aldehyde dehydrogenase) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to its corresponding acid, the osmoprotector glycine betaine. This reaction is involved in the catabolism of choline and in the response of this important pathogen to the osmotic and oxidative stresses prevalent in infection sites. The crystal structure of PaBADH in complex with NADPH showed a novel covalent adduct between the C2N of the pyridine ring and the sulfur atom of the catalytic cysteine residue, Cys286. This kind of adduct has not been reported previously either for a cysteine residue or for a low-molecular-mass thiol. The Michael addition of the cysteine thiolate in the ‘resting’ conformation to the double bond of the α,β-unsaturated nicotinamide is facilitated by the particular conformation of NADPH in the active site of PaBADH (also observed in the crystal structure of the Cys286Ala mutant) and by an ordered water molecule hydrogen bonded to the carboxamide group. Reversible formation of NAD(P)H–Cys286 adducts in solution causes reversible enzyme inactivation as well as the loss of Cys286 reactivity towards thiol-specific reagents. This novel covalent modification may provide a physiologically relevant regulatory mechanism of the irreversible PaBADH-catalysed reaction, preventing deleterious decreases in the intracellular NAD(P)+/NAD(P)H ratios.

Reaction of the active site of papain with a reporter group labeled phenacyl halide

1970 ◽  
Vol 92 (23) ◽  
pp. 6980-6982 ◽  
Author(s):  
Emil T. Kaiser ◽  
Richard W. Furlanetto
Keyword(s):  
Active Site ◽  
Reporter Group

scholarly journals High concentrations of aldehydes slow the reaction of cytoplasmic aldehyde dehydrogenase with thiol-group modifiers

1985 ◽  
Vol 228 (3) ◽  
pp. 765-767 ◽  
Author(s):  
T M Kitson
Keyword(s):  
Binding Site ◽  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Thiol Group ◽  
High Concentrations ◽  
Hydrolysis Of

High concentrations of aldehydes slow the inactivation of cytoplasmic aldehyde dehydrogenase by disulfiram and also slow the reaction of the enzyme with 2,2'-dithiodipyridine. It is concluded that a low-affinity aldehyde-binding site is probably the site at which thiol-group modifiers react with aldehyde dehydrogenase, as well as being the active site for hydrolysis of 4-nitrophenyl acetate.

Enhancement in the Catalytic Activity of Human Salivary Aldehyde Dehydrogenase by Alliin from Garlic: Implications in Aldehyde Toxicity and Oral Health

2019 ◽  
Vol 20 (6) ◽  
pp. 506-516 ◽  
Author(s):  
Amaj A. Laskar ◽  
Danishuddin ◽  
Shaheer H. Khan ◽  
Naidu Subbarao ◽  
Hina Younus
Keyword(s):  
Catalytic Activity ◽  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Bioactive Compound ◽  
Docking Study ◽  
Initial Reaction ◽  
Active Site Residues ◽  
Binding Region ◽  
Binding Studies ◽  
Molecular Docking Study

Background: Lower human salivary aldehyde dehydrogenase (hsALDH) activity increases the risk of aldehyde mediated pathogenesis including oral cancer. Alliin, the bioactive compound of garlic, exhibits many beneficial health effects. Objective: To study the effect of alliin on hsALDH activity. Method: Enzyme kinetics was performed to study the effect of alliin on the activity of hsALDH. Different biophysical techniques were employed for structural and binding studies. Docking analysis was done to predict the binding region and the type of binding forces. Results: Alliin enhanced the dehydrogenase activity of the enzyme. It slightly reduced the Km and significantly enhanced the Vmax value. At 1 µM alliin concentration, the initial reaction rate increased by about two times. Further, it enhanced the hsALDH esterase activity. Biophysical studies indicated a strong complex formation between the enzyme and alliin (binding constant, Kb: 2.35 ± 0.14 x 103 M-1). It changes the secondary structure of hsALDH. Molecular docking study indicated that alliin interacts to the enzyme near the substrate binding region involving some active site residues that are evolutionary conserved. There was a slight increase in the nucleophilicity of active site cysteine in the presence of alliin. Ligand efficiency metrics values indicate that alliin is an efficient ligand for the enzyme. Conclusion: Alliin activates the catalytic activity of the enzyme. Hence, consumption of alliincontaining garlic preparations or alliin supplements and use of alliin in pure form may lower aldehyde related pathogenesis including oral carcinogenesis.

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