bind carbonic anhydrase with only one point through the cofactor zinc ion and react to form bicarbonate or the reversible reaction [10]. Another example for small molecule is the binding of a covalent adduct formed between pyruvate and nicotinamide adenine dinucleotide (NAD+) to lactate dehydrogenase (LDH) to produce lactate in which only two binding points, namely, the carbonyl group and the carboxylate group, of pyruvate are used to bind with the LDH [12]. The number of binding sites of an enzyme to the substrate is important in determining the type of enzyme specificity. The molecular recognition for enzyme specificity has been categorized into three major types of specificities: substrate specificity, regiospecificity, and stereospecificity [13].
1.4.1 Substrate Specificity
As a catalyst, the most distinguished property of enzyme is its substrate specificity. This kind of specificity determines its unique biological function. Many enzymes catalyze only one particular biological substrate. Enzymes with this kind of specificity are, therefore, called having absolute substrate specificity. For example, glucose‐6‐phosphatase binds only glucose‐6‐phosphate (G6P) to hydrolyze catalytically the phosphate moiety from the G6P. Therefore, the substrate specificity of glucose‐6‐phosphatase is absolute substrate specificity. Other enzymes possess a broader substrate specificity called relative group specificity that takes a group of biological molecules of similar chemical structure as substrate. As an example, acid and alkaline phosphatase can form phosphate ester bond for a variety of substrates. Therefore, acid and alkaline phosphatase presents wider relative group specificity.
Whether an enzyme has absolute substrate specificity or relative group specificity depends on the number of binding sites of the enzyme–substrate complex. Figure 1.1 illustrates the number of binding sites of an enzyme to the substrate makes the enzyme absolute substrate specificity or relative group substrate specificity. There is only one specific molecule that can attach the enzyme with four binding sites as shown in Figure 1.1a that gives absolute substrate specificity for the enzyme. The number of biding sites for the enzyme with substrate shown in Figure 1.1a–c is three, two, and one, respectively, that makes a variety of different groups available for the unbound group in the substrate to produce a relative group specificity for the enzyme. The substrate specificity of enzyme brings the advantage of reducing the possible side reactions, thus eliminating the laborious purification and separation works.
Figure 1.1 The number of binding sites of a substrate with enzyme determines the type of enzyme specificity: (a) Absolute substrate specificity of enzyme, (b–d) relative group specificity of enzyme.
1.4.2 Regiospecificity
Some of the enzymes may selectively catalyze one functional group at certain region of the molecule among several similar functional groups located at different regions of the same molecule. This kind of substrate specificity is called regiospecificity or diastereospecificity [13]. Example of regiospecificity in organic synthesis has been found since 1986 that the regioselective deacylation of methyl 2,3,4,6‐tetra‐O‐acyl‐D‐hexopyranosides gives the 6‐OH derivatives in high yields using the lipase from Candida cylindracea [14]. The enzyme‐catalyzed regioselective O‐acylation of ribo‐, arabino‐, xylo‐, rhamnopyranosides, and aryl pyranosides is reviewed, and the methodology is applied to the total synthesis of the naturally occurring rhamnopyranoside by Bashir et al [15]. Regioselective biotransformation of dinitrile compounds 2‐, 3‐, and 4‐(cyanomethyl) benzonitrile can be performed by whole bacterium cell Rhodococcus rhodochrous to the corresponding 2‐(cyanophenyl) acetic acid, 3‐ or 4‐(cyanomethyl) benzoic acid with high yield [16]. Whole cell bacterium Bacillus cereus has also been used for regioselectively converting 2‐phenylenediamine to 2‐aminoacetanilide with a76% molar yield [17]. Other than bacterium, monooxygenase in the phytopathogenic fungi Colletotrichum gloeosporioides and Botrytis cinerea has been found having the ability of regioselective hydroxylation of the C‐H bonds to yield the corresponding diols [18]. Enzyme in cultured plant cells of Phytolacca Americana can reduce, and regioselectively hydroxylate and glucosylate, raspberry ketone and zingerone to their β‐glycosides [19]. Ginkgo biloba cell suspension cultures were used to regio‐ and stereoselectively convert sinenxan A, 2α,5α,10β,14β‐tetra‐acetoxy‐4(20), 11‐taxadiene, a taxoid isolated from callus tissue cultures of Taxus spp., in Taxol® synthesis [20]. The regioselective oxidation of (–)‐verbenone, an important component of the essential oil from rosemary, to (–)‐10‐hydroxyberbenone with human liver microsomes has been investigated by Miyazawa et al [21]. Although regioselective oxidation of terpenoids is difficult by chemical methods, regioselective oxidation of (+)‐ and (–)‐citronellene was recently performed with Spodoptera litura, a larvae of common cutworm, to (2R,3S)‐3,7‐dimethyl‐6‐octene‐1,2‐diol (yield: 89.7%) and (2S,3R)‐3,7‐dimethyl‐6‐octene‐1,2‐diol (yield: 56.3%) [22]. These examples of enzyme‐catalyzed regiospecificity clearly elucidates that the specific enzyme–substrate binding configuration at the active site allows only one of several similar function groups in different regions of the substrate molecule reacts to produce the product. The number of binding points of enzyme–substrate complex for regioselective molecular recognition must be a “multi‐point” binding case to match the complexity of the substrate molecule.
1.4.3 Stereospecificity
The most magnificent specificity of enzyme is its distinguishable ability for only one enantiomeric structure of racemic substrate molecules. The molecular recognition of an enzyme for enantiomeric molecules is called enantiospecificity or stereospecificity. The stereospecificity is an intrinsic property of enzyme which is due to the chirality of active site of the enzyme. Except for a few cases, all enzymes are chiral catalysts because they all made from L‐amino acids, thus the binding of asymmetric substrate at the active site is stereoselective. Since the stereospecificity of enzyme involves enzyme–substrate complex formation with only one enantiomer of a racemate, only one product is formed from the enzyme‐catalyzed reaction. Therefore, enzyme‐catalyzed reactions are in great favor of the organic asymmetric synthesis [23].
The theoretical explanation for the stereospecificity of enzyme was based on the rationale of three‐point attachment rule [24, 25]. This rule suggests that at least three different binding points should occur between enzyme and substrate at the active site to make recognition for the correct stereostructure of substrate molecule as illustrated in Figure 1.2. However, the three‐point attachment is not strict for the stereospecificity of enzyme to asymmetric molecules. As shown in Figure 1.3, the recognition of the stereostructure of asymmetric molecules with enzyme can be accessed by two‐point binding of enzyme–substrate complex, under the situations that the stability of the enzyme–substrate complex is greatly influenced by the formation of two different 3D tetrahedral structure of enzyme–substrate complex or the ability of interaction for the two unbinding groups of the substrate with enzyme at the active site is obviously different. With only two‐point binding of the enzyme–substrate complex, one of the two substrate enantiomers possessing greater stability of the enzyme–substrate complex will have enough time to react to form product, but this situation will not happen for the other substrate enantiomer.
The preparation of enantiopure compounds is highly demanded by industries, particularly, in pharmaceutical