Stereochemical specificity, of enzymes

Problem 21.51 Account for the stereochemical specificity of enzymes with chiral substrates. 

The stereochemical specificity of enzymes depends on the existence of at least three different points of interaction, each of which must have a binding or catalytic function. A catalytic site on the molecule is known as an active site or active centre of the enzyme. Such sites constitute only a small proportion of the total volume of the enzyme and are located on or near the surface. The active site is usually a very complex physico-chemical space, creating micro-environments in which the binding and catalytic areas can be found. The forces operating at the active site can involve charge, hydrophobicity, hydrogen-bonding and redox processes. The determinants of specificity are thus very complex but are founded on the primary, secondary and tertiary structures of proteins (see Appendix 5.1). 

When a racemic substance is hydrogenated or when the reduction leads to the production of centers of asymmetry, the phytochemical reduction will take at first a completely or partially asymmetric course. Examples of such asymmetric reactions are the conversions of pure racemic valeraldehyde, acetaldol, furoin and furil, diacetyl and acetyl-methylcarbinol to optically active alcohols. Occasionally meso forms also arise, as for example in the case off glycols (p. 84). The reasons for the stereochemical specificity of these reactions have not been clarified. This type of phenomenon has frequently been observed in the related intramolecular dismutation of keto aldehydes, especially if enzyme materials of differing origins are used. 

Complexation could occur in many different ways, but for the intimate com-plexation required for catalysis, the enzyme must have, or must be able to assume, a shape complementary to that of the substrate. Originally, it was believed that the substrate fitted the enzyme somewhat like a key in a lock this concept has been modified in recent years to the induced-fit theory, whereby the enzyme can adapt to fit the substrate by undergoing conformational changes (Figure 25-18), Alternatively, the substrate may be similarly induced to fit the enzyme. The complementarity is three-dimensional, an important factor in determining the specificity of enzymes to the structure and stereochemical configuration of the substrates. 

Further progress on the problem of the stereochemical specificity of cyclitol oxidation by A. suboxydans will depend on the isolation of the enzyme or enzymes involved. Cell-free preparations capable of oxidizing wn/o-inositol have been obtained,43 44 but these have not been further purified. The enzyme is apparently a true dehydrogenase, since it can couple with diaphorase.44... 

It is probably as stable probes of the specificity of enzymes that carba-sugars find their justification. A striking example of their utility is illustrated by the elucidation of the stereochemical selectivity of the reverse reaction of cellobiose phosphorylase, where only the 5a-carba-(3-D-glucopyranose was a substrate of the reaction — not the a anomer (Scheme 8.2) [11]. 

Enzymes are also classified on the basis of their specificity. The four classifications of specificity are absolute, group, linkage, and stereochemical specificity. An enzyme with absolute... 

The importance of obstruction (steric hindrance) in determining the stereochemical course of enzymic reactions is further illustrated by a comparison of the stereospecificities of the alcohol dehydrogenases from yeast and from horse liver. The enzymes from both sources are A-side (re face) specific dehydrogenases wherein the pro-(R) hydrogen at C-l of the alcohol is in the transferring position Eq. (2)] ... 

Stereochemical specificity. Some enzymes catalyze reactions of only one stereoisomer of a compound. 

One outstanding feature of enzymes is that many of them are highly specific. Those that act on carbohydrates are particularly so, the slightest change in the stereochemical configuration of the molecule being sufficient to make a particular enzyme incompatible and unable to elTect hydrolysis. 

The characteristics of enzymes are their catalytic efficiency and their specificity. Enzymes increase the reaction velocities by factors of at least one million compared to the uncatalyzed reaction. Enzymes are highly specific, and consequendy a vast number exist. An enzyme usually catalyzes only one reaction involving only certain substrates. For instance, most enzymes acting on carbohydrates are so specific that even the slightest change in the stereochemical configuration is sufficient to make the enzyme incompatible and unable to effect hydrolysis. 

Stereochemical probes of the specificity of substrates, products, and effectors in enzyme-catalyzed reactions, receptor-ligand interactions, nucleic acid-ligand interactions, etc. Most chirality probe studies attempt to address the stereospecificity of the substrates or ligands or even allosteric effectors. However, upon use of specific kinetic probes, isotopic labeling of achiral centers, chronfium-or cobalt-nucleotide complexes, etc., other stereospecific characteristics can be identified, aU of which will assist in the delineation of the kinetic mechanism as well as the active-site topology. A few examples of chirality probes include ... 

NADH. These experiments were pioneering with respect to contemporary enzymology, especially with regard to early recognition that coenzymes are held within enzyme active sites in stereochemically preferred ways. One typically utilizes NADH that contains a tritium or deuterium atom in the 4R or 45 position, and the success or failure of substrate deuteration/tritiation indicates the stereochemistry. Westheimer has tabulated the known examples of dehydrogenases that exhibit specificity for a particular face of NADH. Creighton and Murthy have reproduced this tabulation in their comprehensive review on the stereochemistry of enzyme-catalyzed reactions at carbon. 

Transketolase catalyzes the reversible transfer of a hydroxyacetyl fragment from a ketose to an aldehyde. Because the ketose products formed by transketolase reactions are not acceptors for a consecutive transformation by the same enzyme, we have investigated the option to include a xylose (glucose) isomerase (Xyll E.C. 5.3.1.5), which has similar stereochemical specificity, for ketose to aldose equilibration (Scheme 2.2.5.13). Starting from racemic lactaldehyde 32a, the transketolase forms 5-deoxy-D-xylulose 35a, which indeed was accepted by the Xyll in situ for diastereospecific conversion into 5-deoxy-D-xylose 36a. The latter again proved to be a substrate of transketolase which completed a tandem operation to furnish 7-deoxy-sedoheptulose 37a as the sole bisadduct in 24% overall yield and in enantio- and diastereomerically pure quality [35, 36]. All four stereocenters of the resulting product are completely controlled by the enzymes during this one-pot operation. The procedure profits from the limited tolerance of the isomerase... 

---