Enzyme prochiral centers

It is generally beheved that selectivity of hydrolytic enzymes strongly depends on the proximity of the chiral center to the reacting carbonyl group, and only a few examples of successful resolutions exist for compounds that have the chiral center removed by more than three bonds. A noticeable exception to this rule is the enantioselective hydrolysis by Pseudomonasfluorescens Hpase (PEL) of racemic dithioacetal (5) that has a prochiral center four bonds away from the reactive carboxylate (24). The monoester (6) is obtained in 89% yield and 98% ee. 

It should be mentioned that the central carbon atom of citric acid becomes chiral when the two peripheral carboxy groups are substituted differently (examples will be found below). For enzyme reactions it is a prochirality center. This has been shown for vibrioferrin (58) and staphyloferrin B (59). 

Most enzymes possess an infallible ability to recognize the difference between the right side and the left side of an organic substrate even when the latter has perfect bilateral symmetry. In fact, this ability is limited to prochiral centers of molecules and is a natural consequence of their reaction with the chiral... 

Another example is provided by malic acid, a chiral molecule which also contains a prochiral center (see Eq. 9-74). In this case replacement of the pro-R or pro-S hydrogen atom by another atom or group would yield a pair of diastereoisomers rather than enantiomers. Therefore, these hydrogen atoms are diastereotopic. When L-malic acid is dehydrated by fumarate hydratase (Chapter 13) the hydrogen in the pro-R position is removed but that in the pro-S position is not touched. This can be demonstrated by allowing the dehydration product, fumarate, to be hydrated to malate in 2HzO (Eq. 9-74). The malate formed contains deuterium in the pro-R position. If this malate is now isolated and placed with another portion of enzyme in H20, the deuterium is removed cleanly. The fumarate produced contains no deuterium. 

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... 

The prochiral center in citric acid is attached to two CH2COOH groups. The configurational question can, therefore, be phrased as the question, does acetyl-CoA contribute the pro-R or pro-S CH2COOH groups To provide an answer proved more difficult than for the relatively simple CabH2H cases so far discussed. The proof requires knowledge about several other enzymes and, as well, information about the chirality of compounds related to shikimic acid. 

An additional method to determine the nature of the metal-nucleotide complex that is preferred by the enzyme is in the study of the effects of cation substitution on the stereoselectivity of chiral-specific thiophosphate nucleotides. Thiophosphate derivatives of cAMP, AMP, ADP, and ATP at the a-, /3-, and/or y-positions were initially introduced by Eckstein and co-workers (66, 67) and expanded to several other nucleotides. Substitution of a phosphate oxygen by sulfur at the a-position of cAMP, ADP, or ATP or at the /3-position of ATP gives rise to a new chiral center, Substitution in the a-position of AMP, j8-position of ADP, and the y-position of ATP changes an achiral center to a prochiral center. This substitution gives rise to a small decrease in p a for the phosphate, and with many enzymes these thiophosphate analogs have a decreased substrate ac-... 

The coordination structures of enzyme-bound manganese nucleotides can in favorable cases be determined by analysis of electron paramagnetic resonance (EPR) spectra of Mn(II) coordinated to O-labeled nucleotides. When the nucleotide is stereospecifically labeled with O at one diastereotopic position of a prochiral center, either oxygen can in principle be bound to Mn(II) in the coordination complex in an enzymic site. When the coordination bond is between Mn(II) and O, the EPR signals for Mn(II) are broadened and attenuated, owing to unresolved superhyperfine coupling between the nucleus of 0 and the unpaired electrons of Mn(II) (23). No such effect is possible with 0, which has no nuclear spin. The effect is observable in samples in which all the Mn(Il) is specifically bound in one or two defined complexes of the nucleotide with the enzyme. Thus the complex Mg(Sp)-[a- 0]ADP bound at the active site of ere-... 

In this review, we shall concentrate on the stereochemistry of enzymic reactions of amino acids, many of which involve transformations at prochiral centers. We shall use the nomenclature of Hanson (8) to specify the stereochemistry of prochiral atoms and groups as pro-R (Hjj) and pro-S (Hj) and of prochiral faces as Re and Si and the nomenclature of Mislow and Raban (2) to describe prochiral groups as having enantiotopic or diastereotopic relationships. Reviews on the stereochemistry of enzymic reactions of amino acids were published in 1978 (9,10), and since the seminal review by Dunathan in 1971 (11), several reviews comparing the stereochemistry of pyridoxal phosphate-catalyzed enzymic reactions have appeared (12-15). 

This method, based on the use of microbial cells or enzymes, exploits three important features of biocatalysts (1) directing a reaction exclusively toward one enantiomer, (2) transforming a prochiral center to a chiral product, and (3) carrying out transformations on nonfunctionalized centers. The traditional problem associated with the enzymatic method is the presumption that these reactions should, of necessity, be carried out in dilute aqueous solutions to mimic biological systems. This leads to problems such as expensive separations and sensitivity of fermentations to deactivating influences. Despite these limitations, the biochemical route offers an attractive alternative for synthesizing an enantiomer directly (Knowles, 1986 Sheldon, 1996). 

Enzyme-catalyzed asymmetric syntheses involve two types of reactions (1) the asymmetric reduction of a prochiral center and (2) the resolution of a racemic material by selective reaction of one enantiomer. Both types arc demonstrated in the syntheses of chiral insect phermones reviewed by Sonnet (1988). Enzymes that have broad substrate specificity and still retain other selectivity features can be versatile and powerful catalysts. In addition, enzyme catalysis is applicable not only in aqueous media but also in nonaqueous solvents, including supercritical fluids (20-22), In all cases, however, enzymes require water to function as catalysts. A small amount of water, corresponding to a monolayer on the enzyme molecule, is usually sufficient (20),... 

The desymmetrization of some prochiral dithioacetal esters possessing up to five bonds between the prochiral center and the ester carbonyl - the site of reaction -proceeded with high selectivity using PSL [454], This example of a highly selective chiral recognition of a remote chiral/prochiral center is not unusual amongst hydrolytic enzymes [455 57]. 

You might note that C2 of glycerol is a prochiral center with two identical arms, a situation similar to that of citrate in the citric acid cycle (Section 22.4). As is typical for enzyme-catalyzed reactions, the phosphorylation of glycerol is selective. Only the pro-R arm undergoes reaction, although this can t be predicted in advance. 

The substrate here has a prochiral center and one hydrogen is transferred specifically only from one face (the re-face) of the double bond to the carbonyl function. It is primarily the chirality of the enzyme which determines the correct course of the reaction. Another example of this is when these same two substrates are in the presence of the enzyme aldolase, to give fructose diphosphate, the Hg rather than the Hg-hydrogen is exchanged with water. 

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