Aldehyde dehydrogenases, active site thiols

Figure 2. Illustration of the importance of the choice of reaction conditions on the determination of initial velocity. Shown are four conditions applied to examine the rate behavior of Escherichia coli NAD+-dependent coenzyme A-linked aldehyde dehydrogenase (Reaction NAD+ + CoA-SH + Acetaldehyde = NADH + Acetyl-S-CoA + H+). All assay mixtures contained enzyme, 0.5 mM NAD+, 8 /jlW CoA-SFI, 16 mM acetaldehyde, and 22.5 mM Tris buffer at pFI 8.1. (a) Time-course observed when enzyme was added to the standard assay (b) time-course observed when enzyme was added to standard assay augmented with 10 mM 2-mercaptoethanol (c) time-course observed when enzyme was first preincubated for 15 min with 8 /jlW CoA-SH, 16 mM acetaldehyde, 10 mM 2-mercaptoethanol, and 22.5 mM Tris buffer at pH 8.1, and the reaction was initiated by addition of NAD+ (d) time-course observed when enzyme was preincubated with lOmM 2-mercaptoethanol for 15 min andthen addedtostandard assay augmented with 10 mM 2-mercaptoethanol. The data are most compatible with the idea that the enzyme has an active-site thiol group that must be reduced to express full catalytic activity during assay.

The catalysis of the oxidation of aldehydes to carboxylates by alcohol dehydrogenases raises questions regarding the function of the active site thiols found in most aldehyde dehydrogenases. Clearly a free thiol is not mechanistically essential for aldehyde oxidation. For example, pig heart lactate dehydrogenase catalyzes the facile oxidation of glyoxalate to oxalate (71), glucose-6-... 

Though the symptoms of the biological action of coprine and disulhram are similar, it was demonstrated that the mechanisms of action are different. Contrary to cyclopropanone hydrate, coprine inhibits mouse liver aldehyde dehydrogenase only in vivo but not in vitro. Based on this observation Wiseman and Abeles (429) assumed that coprine itself is inactive in vivo but is activated by hydrolysis to give initially cyclopropanone hemia-minal and ultimately cyclopropanone hydrate. After enzymatic dehydration to cyclopropanone, this compound forms a kinetically stable thiohe-miketal with the enzyme active-site thiols, leading to inactivation of aldehyde dehydrogenase in the enzyme-catalyzed oxidation of acetaldehyde to acetic acid (Scheme 97). 

Most inhibitors of aldehyde dehydrogenase are inhibitors because they react with the thiol group at the active site of the enzyme. Inhibitors such as disulfiram (Fig. 4.31) have been used in the treatment of alcoholism because if someone drinks alcohol while taking the inhibitor, there is a buildup of acetaldehyde, which causes many unpleasant symptoms such as flushing and nausea (77,78). However, if someone drinks a large amount of alcohol while taking disulfiram it can lead to a life-threatening reaction. 

Several different amino acid side chains can act as nucleophiles in enzyme catalysis. The most powerful nucleophile is the thiol side chain of cysteine, which can be deproto-nated to form the even more nucleophilic thiolate anion. One example in which cysteine is used as a nucleophile is the enzyme glyceraldehyde 3-phosphate dehydrogenase, which uses the redox coenzyme NAD+. As shown in Fig. 10, the aldehyde substrate is attacked by an active site cysteine, Cys-149, to form a hemi-thioketal intermediate, which transfers hydride to NAD+ to form an oxidized thioester intermediate (7). Attack of phosphate anion generates an energy-rich intermediate 3-phosphoglycerate. 

Fig. 13. Aldehyde dehydrogenases utilize thiol-mediated reactions in either of two ways. The top pathway demonstrates the capture of the energy of aldehyde oxidation through formation of a car-boxylate-phosphoric acid anhydride, which can subsequently generate ATP. The bottom pathway shows the hydrolysis of the active site thioester to the thermodynamically favored carboxylate.

Fig. 19. The traditional function for a thiol in an active site is shown in (a). The following alternative functions are illustrated, (b) Prevention of aldehyde reduction in the presence of bound NADH by thiohemiacetal formation, (c) Steric exclusion of secondary alcohols to prevent activity as a secondary alcohol dehydrogenase, (d) Formation of an intimate interaction between the thiol and NAD+ such as occurs in G3PDH to prevent functioning as an alcohol dehydrogenase.

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