Acetoacetate decarboxylase

By far the most well studied enzyme catalyzing a decarboxylation reaction in which iminium ion formation is involved, is acetoacetate decarboxylase [79]. This enzyme obtained from Clostridium acetobutylicium [80] has an apparent molecular weight of approximately 340000, and is composed of subunits with a molecular weight of 29000 [81], It is apparently composed of 12 subunits, and these may be dissociated 

The cleaver introduction of a reporter group was used to determine the pK of the 

The decarboxylation of y8-keto acids by both primary amines and by acetoacetate decarboxylase has been studied by the method of carbon isotope effects [90,91]. A typical isotope effect for the amine-catalyzed reaction is 1.03 for the cyanomethylamine-catalyzed decarboxylation of acetoacetate at pH 5.0. This demonstrates that the carbon-carbon bond is being cleaved in the rate-determining decarboxylation step. The comparable carbon isotope effect for the enzymatic decarboxylation is = 1.018 and is pH-independent over the range pH 

This data suggests that in the enzymatic reaction the decarboxylation event is at least partially rate-determining. 

A variety of /8-diketones have been demonstrated to be very effective inhibitors of acetoacetate decarboxylase [83]. Although they bind tightly to the active site they are not reduced by borohydride. Corresponding a- or y-diketones are not inhibitory [84]. Acetopyruvate, for example, inhibits competitively with respect to acetoacetate with 

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BOROHYDRIDE REDUCTION ACETAZOLAMIDE Acetic acid, autoprotolysis constant, AUTOPROTOLYSIS ACETOACETATE DECARBOXYLASE Acetoacetate decarboxylase reduction, BOROHYDRIDE REDUCTION ACETOLACTATE SYNTHASE Acetone,... 

In 1962 too, Fridovich showed that the addition of sodium borohydride to a mixture of acetoacetate decarboxylase and acetoacetate inactivates the enzyme, whereas the addition of borohydride to a buffered solution of the enzyme alone has no effect on the rate at which it can promote the decarboxylation of acetoacetate (Fridovich and Westheimer, 1962) this work confirmed the ketimine mechanism that had previously been advanced for the decarboxylation. Subsequent work (beyond the scope of this review) showed that the reaction product, on hydrolysis, yielded e-isopropyllysine [8], formed by the reduction of the ketimine of acetone (11), and control experiments showed that this ketimine was actually an intermediate in the enzymic pathway, as had been postulated (Warren et al., 1966). 

In this system k2 values were easily measured in solutions with large excesses (about 150 1) of catalytic sites over substrate concentrations. Under these conditions the observed first-order rate constant was unaffected by increasing polymer concentration. The variation of k2 over the pH range 3 to 7 is displayed in Fig. 20. It is bell shaped and exhibits a maximum at pH 4.5. This bell-shaped pH-rate profile is similar to that of other model primary amines57,58 63 as well as the enzyme acetoacetate decarboxylase.52... 

The similarity in pH-rate profile for the polymer (Fig. 20) and for simple primary amines or acetoacetate decarboxylase suggests that the mechanistic pathways of the decarboxylation reaction may also be alike. In the model amine and enzyme systems there is good evidence for the pathway shown in (33) ... 

Tin healthy people, acetone is formed in very small amounts from acetoacetate, which is easily de-carboxylated, either spontaneously or by the action of acetoacetate decarboxylase (Fig. 17-18). Because individuals with untreated diabetes produce large quantities of acetoacetate, their blood contains significant amounts of acetone, which is toxic. Acetone is volatile and imparts a characteristic odor to the breath, which is sometimes useful in diagnosing diabetes. ... 

Some decarboxylases do form Schiff bases with their substrates, and some are dependent on metal ions.235 The acetone-forming fermentation of Clostridium acetobutylicum requires large amounts of acetoacetate decarboxylase (Eq. 13-44). 

Decarboxylation. When the amino acid is aspartate, the second compound in equation 2.44 is analogous to the Schiff base in the acetoacetate decarboxylase reaction and may readily decarboxylate ... 

NH2 (lysine) Acetoacetate decarboxylase, aldolase, transaldolase, pyridoxal enzymes Schiff base... 

In biochemical decarboxylation reactions where the reactant contains a 3-keto group, the e-amino group of a lysyl side chain of the protein backbone can form an iminium derivative with the substrate.82 Upon loss of carbon dioxide, the delocalized, weakly basic product will not react faster than carbon dioxide can separate. Benner83 showed that the stereochemical consequence of decarboxylation of acetoacetate by acetoacetate decarboxylase involves protonation of the product from either face, consistent with a passive, uncatalyzed step, which is consistent with the view we have presented. 

Formation of a Schiff Base, Part I Acetoacetate Decarboxylase, Aldolase... 

Aldolases such as fructose-1,6-bisphosphate aldolase (FBP-aldolase), a crucial enzyme in glycolysis, catalyze the formation of carbon-carbon bonds, a critical process for the synthesis of complex biological molecules. FBP-aldolase catalyzes the reversible condensation of dihydroxyacetone phosphate (DHAP) and glyceralde-hyde-3-phosphate (G3P) to form fructose-1,6-bisphosphate. There are two classes of aldolases the first, such as the mammalian FBP-aldolase, uses an active-site lysine to form a Schiff base, whereas the second class features an active-site zinc ion to perform the same reaction. Acetoacetate decarboxylase, an example of the second class, catalyzes the decarboxylation of /3-keto acids. A lysine residue is required for good activity of the enzyme the -amine of lysine activates the substrate carbonyl group by forming a Schiff base. 

R. A. Laursen and F. H. Westheimer, The active site of acetoacetate decarboxylase,... 

Note that the p/C, of a residue at the active site can vary substantially from the value observed for the free amino add in water [27]. For example, the active site of the enzyme acetoacetate decarboxylase features a lysine residue with a very low p/C, of 5.9 (cf. pJCa 9 for the free amino add). In this case, the pICa is influenced by an adjacent protonated lysine residue (Figure 5.6) [28,29]. A similar redudion, though less dramatic, is observed for aliphatic diamines (e.g., the two p/C, values for 1,4-diaminobutane are 10.80 and 9.35, respectively). 

Figure 5.6 A protonated lysine residue at position 116 facilitates the low p/

Kokesh, F.C. and Westheimer, F.H. (1971) Reporter group at the active site of acetoacetate decarboxylase. II. Ionization constant of the amino group./. Am. Chem. Soc., 93, 7270. 

Highbarger, L.A., Gerlt, J.A. and Kenyon, G.L. (1996) Mechanism of the reaction catalyzed by acetoacetate decarboxylase. Importance of lysine 116 in determining the pFQ of active-site lysine 115. Biochemistry, 35, 41. 

Kresge, A.J. (1997) Electrostatic effects on acid dissociation constants. Importance of lysine 116 in determining the p /<, of lysine 115 in the active site of acetoacetate decarboxylase. Chemtracts, 10, 27. 

Investigations show that the active amino group of acetoacetate decarboxylase—the one that is concerned with the formation of the ketimine intermediate—has an especially low pK (26, 27). Model experiments revealed that amines of low pK are the best nonenzymic catalysts in particular, cyanomethylamine led to a rate of decarboxylation that is only a few orders of magnitude less than that for the enzymic system (28, 29, 30). 

Primary amine catalysis (usually involving a lysine residue) has been recognised to play an important role in various enzyme-catalysed reactions. Examples are the conversion of acetoacetate to acetone catalysed by acetoacetate decarboxylase, the condensation of two molecules of S-aminolevulinic acid catalysed by -aminolevulinic deshydratase during the biosynthesis of porphyrins, and the reversible aldol condensation of dihydroxy-acetone phosphate with glyceraldehyde which in the presence of aldolase yields fructose-1-phosphate (64) (For reviews see, for example, Snell and Di Mari,... 

Shemin, 1972 Horecker et al., 1972). In these enzymatic reactions, the mechanism involves initial SchifFs base (imine) formation and subsequent tautomerisation leading to the enamine (C02 elimination in the process of acetoacetate decarboxylase). 

Acetone was produced during World War I by using engineered strains of Clostridium bacteria. These strains make an enzyme called acetoacetate decarboxylase that catalyzes the decarboxylation of acetoacetate. 

The KIEs for scission of the C—C bond to form the enamine (by measuring 45C02/44C02 ratios) of both the model amine and the acetoacetate decarboxylase catalyzed reactions were studied by O Leary and Baughn125. In the primary amine catalyzed reaction the KIE exhibited pH-dependence consistent with the kinetic data above. In the enzyme catalyzed reaction a pH independent k12/k13 KIE of 1.018 was reported, consistent with the idea that a nucleophilic attack by the lysine amino group and decarboxylation are both partially rate-limiting. 

Schmidt, D.E., and Westheim, F., 1971, Pk of lysine amino group at active site of acetoacetate decarboxylase. Biochemistry 10 1249-1253. 

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