Thiohemiacetal

FIGURE 19.18 A mechanism for the glycer-aldehyde-3-phosphate dehydrogenase reaction. Reaction of an enzyme snlfliydryl with the carbonyl carbon of glyceraldehyde-3-P forms a thiohemiacetal, which loses a hydride to NAD to become a thloester. Phosphorolysls of this thloester releases 1,3-blsphosphoglycerate. 

As in the oxidation of alcohols, the reaction involves the loss of two hydrogen atoms rather than the addition of an oxygen atom. The mechanism of the oxidation mediated by aldehyde dehydrogenase is similar to that of ALD, but first the enzyme must form a thiohemiacetal with the substrate to facilitate the loss of hydride (76) as illustrated in the following reaction sequence (Fig. 4.30). 

The second method consists in the reaction of 3-mercaptopropionic acid amides with various aldehydes and ketones (method B) this reaction proceeds via the formation of thiohemiacetal 143. Method B was modifed (in the case of p-chlorobenzaldehyde) by means of dithioacetal 144, which was isolated and then introduced into the reaction with the second mole of aldehyde (Scheme 49). 

Reaction evidently involves formation of the S-thiohemiacetal with the super-reactive Cys-149. His-176 is thought to activate Cys-149 and to facilitate proton removal (the oxidation step being transfer of hydride to the coenzyme, with formation of the thioacyl intermediate). Thr-179 and residue 181 (e.g., Thr or Asn) probably interact with the 3-phosphate, while Ser-148 can possibly form hydrogen bonds with the C-2 hydroxyl and with the inorganic phosphate (Fig. 19B) [52]. Thr-208 or Arg-231 may also bind the inorganic phosphate. Nucleophilic attack by the phosphate on C-l, with rupture of the C-S bond, gives 1,3-diphosphoglycerate. 

One of the key intermediates shown in this reaction scheme is the formation of a tetrahedral adduct during acylation and deacylation (84). Additional support for the formation of a tetrahedral intermedite comes from the observation already referred to— that aldehydes may act as potent inhibitors of papain. Westerik and Wolfenden (65) attribute the inhibitory eflFect of aldehydes to the formation of a stable thiol adduct (thiohemiacetal) analogous to the tetrahedral intermediate produced when papain acts on a substrate. This relationship is depicted in Figure 14. When the complete picture for the mechanism of catalysis by the thiol proteases finally emerges, it will no doubt be similar to the mechanism of action of the serine proteinases. 

In this mechanism, the thiol group of cysteine is used both as catalyst and to preserve and trainsfer the free energy of the oxidation reaction. Thus, the carbon of the thiohemiacetal is less (+) than an acetal carbon and so it is easier to remove a hydride ion using NAD+, and the resulting thiol ester is a high energy compound which is readily attacked by phosphate. 

The addition of a range of thiols to cyclopropanone at — 30 °C occurred smoothly to afford the thiohemiacetals 5, which are useful precursors to 1-halocyclopropyl alkyl sulfides 6 and 7, formed upon reaction with anhydrous hydrogen bromide or hydrogen chloride. ... 

Glyceraldehyde 3-phosphate is oxidized by NAD+, and inorganic phosphate (Pi) is incorporated into the product to form an acyl phosphate, 1,3-bisphosphoglycerate. NAD+ is reduced by transfer of a hydride ion (H ) from thiohemiacetal to the fourth position on the nicotinamide ring of NAD. ... 

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.

Cys-174, one of the ligands to zinc, has been proposed to form a thiohemiacetal intermediate with the histidinal however, catalysis would require functioning of the enzyme with an altered metal coordination. It is difficult to imagine how activity would be maintained if such a major reorganization were to occur. 

E. coli has been characterized in vitro and the reaction proceeds through a covalent thiohemiacetal intermediate bound at Cysl49." The enzyme is a tetramer with a subunit mass of 37 kDa. A of 20 s, a... 

Kozarich and Chari suggest that the enzymic reaction most likely involves a cis-enediol intermediate, on the basis of two observations. First, the enzyme has the surprising ability to catalyze the stereospecific conversion of the thiohemiacetal (11), formed from (glutathiomethyl)glyoxal (10) and j8-mercaptoethanoI, to the thioester 12, established to have the (8)-configuration at C-2 on the basis of chemical degradation to L-lactate (111) [Eq. (24)] ... 

Two mechanisms for the NAD -dependent oxidation of an alcohol to a carboxylate have been characterized in enzymatic reactions. In the first mechanism, an active-site cysteine plays a crucial role in the reaction. A hydride is transferred to NAD from the alcohol substrate to generate an aldehyde intermediate, then the cysteine thiolate attacks the aldehyde to form a thiohemiacetal intermediate. The thiohemiacetal is oxidized by the second NAD" " to form a thioester, which is hydrolyzed to generate the carboxylate product. The second mechanism is similar to the first, except that the aldehyde undergoes hydration instead of thiohemiacetal formation. The aldehyde hydrate is oxidized by NAD" " to form the observed product. This reaction proceeds... 

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