Studies of the esterase activity of cytosolic aldehyde dehydrogenase with resorufin acetate as substrate.
Resorufin acetate is a very good substrate for sheep liver cytosolic aldehyde dehydrogenase, both from the point of view of practical spectrophotometry and in terms of information provided about the nature of the catalysis shown by this enzyme. p-Nitrophenyl (PNP) acetate competes against resorufin acetate for the enzyme's active site (although relatively weakly as the latter substrate has the lower Michaelis constant), but acetaldehyde (in the presence of NAD+) inhibits the hydrolysis of resorufin acetate only at very high aldehyde concentration. In the absence of cofactor, the rate-limiting step in the hydrolysis of resorufin acetate and of PNP acetate is hydrolysis of the common acetyl-enzyme, as shown by the observation of bursts of chromophoric product and very similar values of kcat. In the presence of NAD+ or NADH, however, the deacylation step with resorufin acetate is greatly accelerated until acylation seems to become rate-limiting, because no burst is seen under these conditions. Millimolar concentrations of Mg2+ activate the hydrolyis of resorufin acetate both in the presence and absence of cofactors. With both Mg2+ and cofactor the kcat for hydrolysis of resorufin acetate is 30-35 s-1; this is three orders of magnitude higher than the kcat for aldehyde oxidation in the presence of Mg2+, showing that the enzyme's potential catalytic efficency is very much hampered by the slowness with which NADH dissociates from its binding site. The pH profile for the hydrolysis of resorufin acetate in the presence of NAD+ or NADH fits well to a theoretical ionization curve of pKa approx. 8.2; it is suggested that this might belong to the enzyme's putative catalytic residue (Cys-302).
Kitson
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Departments of Chemistry and Biochemistry, Massey University, Private Bag 11222, Palmerston North, New Zealand.
Kitson
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KE
D000444:Aldehyde Dehydrogenase / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D003600:Cytosol / Q000201:enzymology; D004950:Esterases / Q000032:analysis / Q000378:metabolism*; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D010078:Oxazines / Q000378:metabolism*; D012756:Sheep; D013379:Substrate Specificity
D010078:Oxazines; C014180:resorufin; D000444:Aldehyde Dehydrogenase; D004950:Esterases