Citrate, prochirality

Citrate, prochirality of, 1156 Citrate synthase, active site of, 1046 function of, 1045 mechanism of action of, 1043, 1047... 

Elucidating the stereochemistry of reaction at prochirality centers is a powerful method for studying detailed mechanisms in biochemical reactions. As just one example, the conversion of citrate to (ds)-aconitate in the citric acid cycle has been shown to occur with loss of a pro-R hydrogen, implying that the reaction takes place by an anti elimination mechanism. That is, the OH and H groups leave from opposite sides of the molecule. 

Note that the hydroxyl-bearing carbon of citrate is a prochirality center and contains two identical "arms." Because the initial aldol reaction of acetyl CoA to oxaloacetate occurs specifically from the Si face of the ketone carbonyl group, the pro-S arm of citrate is derived from acetyl CoA and the pro-R arm is derived from oxaloacetate. 

Step 2 of Figure 29.12 Isomerization Citrate, a prochiral tertiary alcohol, is next converted into its isomer, (2, 35)-isocitrate, a chiral secondary alcohol. The isomerization occurs in two steps, both of which are catalyzed by the same aconitase enzyme. The initial step is an ElcB dehydration of a /3-hydroxy acid to give cfs-aconitate, the same sort of reaction that occurs in step 9 of glycolysis (Figure 29.7). The second step is a conjugate nucleophilic addition of water to the C=C bond (Section 19.13). The dehydration of citrate takes place specifically on the pro-R arm—the one derived from oxaloacetate—rather than on the pro-S arm derived from acetyl CoA. 

Worthy of note in this reaction is that citrate displays prochirality (see Section 3.4.7). The methylene carbons may be considered prochiral, in that enzymic elimination of a proton is likely to be entirely stereospecific. In addition, the apparently equivalent side-chains on the central carbon are also prochiral and going to be positioned quite differently on the enzyme. This means that only one of these side-chains is involved in the dehydration-rehydration... 

A stereochemical property of compounds arising from the ability of an enzyme s active site to distinguish between two chemically identical substituents covalently bound to a tetrahedral center (usually carbon and, in some cases, phosphorus). Prochirality is also termed prostereoisomerism. The classical example is citrate with its two carboxymethyl group substituents. Likewise, the Cl carbon atom of ethanol has two prochiral hydrogens. See Chirality Chirality Probes... 

FIGURE 2 The prochiral nature of citrate, (a) Structure of citrate (b) schematic representation of citrate X = —OH Y = —COO- Z = —CH2COO-. (c) Correct complementary fit of citrate to the binding site of aconitase. There is only one way in which the three specified groups of citrate can fit on the three points of the binding site. Thus only one of the two —CH2COO groups is bound by aconitase. 

The citrate ion, a very important prochiral metabolic intermediate, has three prochiral centers at C-2, C-3, and C-4, respectively. That at C-3 distinguishes the pro-R and pro-S arms and determines the stereochemical numbering. Citrate containing 14C in the sn-1 position is called s -citrate[l-14C] and is the form of labeled citrate that is synthesized in living cells from oxaloacetate and [l-14C]acetyl coenzyme A (see Fig. 10-6). The first step in the further metabolism of citrate is the elimination of the -OH group from C-3 together with the Hr proton from C-4 through the action of the enzyme aconitate hydratase (aconitase). In this case the proton at C-4 (in the pro-R arm) is selected rather than that at C-2. 

It was not until 1948 that Ogston popularized the concept that by binding with substrates at three points, enzymes were capable of asymmetric attack upon symmetric substrates.d In other words, an enzyme could synthesize citrate with the carbon atoms from acetyl-CoA occupying one of the two -CH2COOH groups surrounding the prochiral center. Later, the complete stereochemistry of the... 

Ogston s contribution led to an interesting extension of concepts concerning stereochemistry of enzyme action. Compounds of the type Ca2bd are termed prochiral, and it is recognized that an enzyme that either synthesizes such a compound or uses it as a substrate nearly always does so stereospecifically. In the case of citrate synthase, for example, it is inherently likely that the planar carbonyl carbon of oxaloacetate lies flat on an enzyme surface and that only one side of the atom is available for attack by acetyl-coenzyme A. 

Ogston offered an explanation, called the three-point attachment proposal, that was to initiate the concept of prochirality. If citrate is represented as a three-dimensional structure (Fig. 12-12), then on the assumption that a three-point attachment to aconitase is necessary for catalysis, it is apparent that citrate can only be accommodated in one orientation. The removal of the elements of water can then only occur from one particular half of the symmetrical molecule. 

In the reaction with aconitase, one citrate methylene was shown to behave as follows one of its prochiral hydrogens is lost to water, while the other becomes the hydrogen on the a-carbon of isocitrate and is removed to form NADPH by isocitrate dehydrogenase. With the two citrate samples, 118 and 119, the observed result shows that the active methylene cannot be at C-4 (both hydrogens are H) and is, therefore, at C-2. Similarly, with the two citrate samples 120 and 121, the active methylene cannot be at C-2, but must be at C-4. The results are only consistent with the conclusion that the methylene acted on by aconitase was... 

If you perform the replacement test to assign pro-R/pro-S prochirality, you will see that the right arm of citrate is pro-R and the product pictured on the right is formed. 

Ogston [20,21], seemingly unaware of the Easson-Stedman model, proposed a similar three-point attachment model to rationalize the observed stereoselectivity in the enzymatic transformation of symmetrical prochiral substrates, e.g., citrate and aminomalonate (Fig. 3) [22]. Similarly, Dalgleish [23], also unaware of the Easson-Stedman model [17], rationalized his observations concerning the resolution of the enantiomers of a number of amino acids on paper chromatography by a three-point attachment. In a subsequent telephone conversation with Bentley [24], Dalgleish stated that he was terribly impressed by the Ogston hypothesis. It is therefore... 

Asymmetrical metabolism of citrate. Although the citrate molecule has perfect bilateral symmetry, it is degraded asymmetric[Pg.688]

Fig. 5. Formula of citrate with prochiral numbering of the carbon atoms.

Diagrammatic representation of prochirality of citrate. (A) Structure of citrate, (B) Three point landing of citrate on the active site of aconitase. Only one of the CHfiOO- groups, the one which is invohmd in binding, is attacked by aconitase. This is why only one form of labeled a-ketoglutarate is produced. [(Ref. Ogston, AG., Nature, 162 963(1948) ... 

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