Figure 1. During transcription (DNA → mRNA), only one strand of DNA (sense strand) is transcribed, that is, used for the synthesis of complementary mRNA. During ribosomal protein synthesis, the latter is translated into an amino acid sequence or the primary protein structure. The rate of translation is also influenced by the concentrations of the amino acid-specific tRNAs. This and the codon usage, the frequency in the use of the different codons for an amino acid, differ from cell to cell. For Tyr as shown in the figure, there are two codons, TAC and TAT.

Figure 2. Change in the tertiary structure of an enzyme due to substrate binding, illustrated for the binding of glucose to hexokinase: (a) without glucose; (b) after binding of glucose(from Darnell, Lodish, and Baltimore, 1986; copyright 1986 Scientific American Books, with permission by W.H. Freeman and Co.).

Figure 3. Conventions used to describe the substrate specificity of enzymes and the binding (S-, S′-) and catalytically active (C) subsites of the active site of the enzyme, illustrated for peptidases (Schechter and Berger, 1967). This can be applied to all enzymes that catalyze the formation or breaking of a covalent bond. The number of subsites involved depends on the size of the molecules involved. The substrate sequence specificity P _i…P ₁ — P ₁′…P _i′ for the hydrolysis by a peptidase, determined by the binding sites S _i…S ₁ S ₁′…S _i′ can be found in the MEROPS database (Rawlings, Barrett, and Bateman, 2010).

Figure 4. Mechanism of the enzyme-catalyzed hydrolysis of peptides and di- and oligosaccharides bound in the binding subsites of the active site by the exoenzymes carboxypeptidase A and glucoamylase.

1. Hydrolysis of C-terminal amino acids by pancreatic carboxypeptidase A. It has a P ₁ specificity for Gly or Phe, and a specificity for amino acids with aliphatic or aromatic side chains. The binding site consists of Arg ₁₄₅ that forms hydrogen and ion–ion bonds with the carboxyl group, and a hydrophobic pocket (Try ₁₉₈, Tyr ₂₄₈, and Phe ₂₇₉) that binds the hydrophobic side chains. The S ₁ binding site is a hydrophobic pocket that has not yet been clearly identified. The catalytically active amino acid residues C are Glu ₂₇₀ that interacts with a bound water molecule, and His ₆₉, Glu ₇₂, and His ₁₉₆ that bind the Zn ²⁺ ion required for the activity. This ion also interacts with O of the peptide bond. The catalytic reaction starts with a proton transfer from the bound H ₂O to Glu ₂₇₀. Then the formed OH ^– attacks C in the peptide bond carbonyl group, yielding a gem-diol intermediate. In the last catalytic reaction step, this proton is transferred to NH of the peptide bond, leading to its cleavage. This is a general acid–general base reaction mechanism (Xu and Guo, 2009; Kilshtain and Warshel, 2009). R ₁ is the P ₁ amino acid side chain; R ₂ is H, amino acid, or peptide acyl group; is the amino acid side chain.
   
2. A two-dimensional representation of the interactions of the amino acid residues in the active site and neighboring residues of glucoamylase from A. niger with bound methyl α-maltoside (bold face). Note that this distorts the distance between neighboring amino acid residues. The catalytically active residues and the bound water molecule are in red and bold face, respectively. The active site is in the shape of a well where only the nonreducing end of a di- or oligosaccharide can be bound at the bottom of the well (lower part of the substrate). In this well, the catalytically active residues Glu ₁₇₉ and Glu ₄₀₀ (numbering for A. niger glucoamylase) are located so that they can act on the first α-(1–4) bond from the nonreducing end, yielding the exo-activity of this enzyme. The tight binding of the substrate by multiple hydrogen bonds is obvious, and is responsible for high specificity. In most fungal glycoamylases, the active site well continues into a O-glycosylated linker between the catalytic domain and a starch binding domain, which allows the catalytic domain to attack a larger area of a starch granule while tethered to its surface (a mechanism similar to that of cellulases) (see Section 2, Figure 5) (adapted from Coutinho and Reilly, 1994; Aleshin et al., 2003).
   
3. Simplified scheme of the mechanism for the hydrolysis of a di- or oligosaccharide catalyzed by glucoamylase (the acid is Glu ₄₀₀ and the base is Glu ₁₇₉, numbering for A. niger glucoamylase). The general acid–general base reaction mechanism starts with a proton transfer from the bound water molecule to Glu ₄₀₀. Then OH ^– acts as a nucleophile on C-1, causing the hydrolysis of the terminal glucose. Finally, the proton of Glu ₁₇₉ is transferred to the leaving mono- or oligosaccharide. In the final step, rapid proton exchange from Glu ₄₀₀ to water, and from water to Glu ₁₇₉, occurs (not shown).
   

Figure 5. Mechanism of the enzyme-catalyzed hydrolysis of oligosaccharides and peptides bound in the binding subsites of the active site by the endohydrolases α-amylase from A. niger and bovine α-chymotrypsin involving the formation of covalent acyl- or glycosyl-enzyme intermediates.

1. Hydrolysis of amylose by α-amylase. Hydrolysis with net retention of configuration is most commonly achieved via a two-step, double displacement mechanism involving a covalent glycosyl-enzyme intermediate, as is shown in the figure. Each step passes through an oxocarbenium ion-like transition state. Reaction occurs with acid/base and nucleophilic assistance provided by two amino acid side chains, Glu ₂₃₀ and Asp ₂₀₆, located 5.5 Å apart. In the first step (often called the glycosylation step), Asp ₂₀₆ residue plays the role of a nucleophile, attacking the anomeric center between two substrate glucosyl units to form a covalent glucosyl-enzyme intermediate. At the same time, Glu ₂₇₀ functions as an acid catalyst and protonates the glycosidic oxygen and the bond is cleaved. The protonated glucosyl unit at subsite +1 leaves the active site while a water molecule moves in. In the second step (known as the deglycosylation step), the glucosyl-enzyme is hydrolyzed by water, with the other residue now acting as a base catalyst deprotonating the water molecule as it attacks. A third conserved residue, a second aspartate (Asp ₂₉₇), binds to the OH-2 and OH-3 groups of the substrate through hydrogen bonds and plays an important role in substrate distortion (van der Maarel et al., 2002). When another glucose molecule, instead of water, enters the active site, a new glycosidic bond, preferentially with an α-(1–6) bond, is formed (Kelly, Dijkhuisen, and Leemhuis, 2009). The pK value of the acid/base group cycles between high and low values during catalysis to optimize it for its role at each step of catalysis. For α-chymotrypsin 2.5b) see next two pages.
   
2. Hydrolysis of peptide bond with the serine endopeptidase α-chymotrypsin. This enzyme catalyzes the hydrolysis of peptide bonds in the polypeptide chain where the S ₁ binding subsite preferably binds aromatic amino acid residues (Tyr, Trp, Phe). It consists of a hydrophobic pocket (Ser ₁₈₉, Ser ₁₉₀, Met ₁₉₂, Trp ₂₁₅, Gly ₂₁₆, Ser ₂₁₇) that can bind the hydrophobic side chains of the P ₁ aromatic amino acids. The binding site includes the negatively charged Asp ₁₀₂, and can therefore not bind unprotected C-terminal amino acids; thus, this enzyme acts as an endohydrolase. It is less specific than the S ₁ binding subsite; the highest specificity is for Leu, Ser, and due to the negative charge it is specific for Lys and Arg. The serine endopeptidases have the catalytic triad Ser ₁₉₅–His ₅₇–Asp ₁₀₂ in the catalytic subsite. The catalytic reactions start with a proton transfer from Ser to His, and from His to Asp in this triad that results in a nucleophilic attack by the serine oxygen on C in the peptide bond carbonyl group of the substrate. This leads to the formation of an acyl-enzyme intermediate and a leaving peptide whose amino group finally binds the proton that was transferred from Ser to His. The acyl-enzyme is then hydrolyzed due a nucleophilic attack by water activated by the catalytic triad, releasing the other part of the original peptide (Berg et al., 2002). Other nucleophiles can also attack the acyl-enzyme; thus, other products can be formed in this kinetically controlled reaction (see Section 6). These condensation products, however, are later hydrolyzed until the equilibrium state has been reached. R ₁ and are amino acid side chains; R ₂ and are protecting groups or other amino acids of a peptide.
   

Figure 6. Location of the strictly conserved, that is, identical amino acid, residues (as balls), with known functions in six penicillin amidases (from E. coli, P. rettgeri, K. cytrophila, A. viscosus, A. faecalis, B. megaterium), in the 3D ribbon structure of PA from E. coli. The 3D structures have been determined for E. coli (PDB 1PNK without and 1GK9 with bound substrate), P. rettgeri (PDB 1CP9), and A. faecalis (PDB 3ML0). The 146 strictly conserved residues were determined by a multiple amino acid alignment based on the primary sequence of the pro-penicillin amidases; the numbering starts from the N-terminal of the A-chain (blue). The homology (% of strictly conserved residues compared with the E. coli enzyme) varies from »30% (A. viscosus) to almost 90% (K. cytrophila). The strictly conserved catalytically active Ser ₂₆₄ ((a) red and (b) green), the N-terminal of the B-chain (green) is formed by proteolytic processing that removes a linker peptide between the A- and B-chains (see Section 4.1). (a) Thirteen strictly conserved residues (brown) are, as expected, located in and around the active site, and two (yellow) around the tightly bound Ca ²⁺ ion (gray) that holds the A- and B-chains together, and also is required for the correct folding, membrane transport, and processing of the pro-enzyme (Brannigan et al., 1995; McDonough, Klei, and Kelly, 1999; Kasche, Galunsky, and Ignatova, 2003, 2005). (b) Strictly conserved eight positively (yellow) and eight negatively (orange) charged amino acid residues are mainly located far from the active site. They stabilize the enzyme at neutral pH by salt bridges, and determine the stability of the enzyme as a function of pH (see Section 10, Figure 28). The other strictly conserved residues are located far from the active site or the Ca ²⁺ binding site. Their importance for the enzyme function and its activity are still unknown.

Figure 7. Free energy diagram for uncatalyzed (dashed line) and enzyme-catalyzed (solid line) reactions involving one binding step and one chemical reaction (see Figure 4). The apparent activation energy of the latter () is much smaller than that for the uncatalyzed reaction (ΔG ^#); ΔG _S is free energy change due to substrate binding.

Figure 8. Free energy diagram for an enzyme-catalyzed reaction involving the formation of an acyl-enzyme intermediate, consisting of one binding process and two chemical reactions as for the peptide or amide bond hydrolysis shown in Figure 5 with either a S- (solid line) or R-amino acid (dashed line) in the position, respectively (see Figure 3). The acyl-enzyme E-A is formed by acylation by the amino acid in the P ₁ position.

Figure 9. Schematic time dependence (a) and experimental results (b) for kinetically and equilibrium-controlled reactions catalyzed by hydrolases with acyl-enzyme and other covalent intermediates.

1. This also shows that the steric purity of the product in equilibrium-controlled racemate resolutions depends on the stereoselectivity of the used enzyme.
   
2. Kinetically (solid line) and equilibrium-controlled (dashed line) synthesis of penicillin G (AN) from equal concentrations of phenylacetyl glycine (AB) or phenylacetic acid (AOH) and 6-aminopenicillanic acid (NH) at different enzyme (penicillin amidase from E. coli) concentrations given in U ml ^–1 (pH 6.0, 25 °C). The kinetically controlled maximum is (within experimental error) independent of the concentration of the enzyme, and is much larger than the equilibrium concentration.
   

Figure 10. Michaelis–Menten plot – that is, rate v (Eq. ( 8)) in units of V _max – for an enzyme-catalyzed reaction as a function of the substrate concentration (in K _m units) at enzyme concentrations [EH] ₀ (lower) and 2[EH] ₀ (upper curve). Note that v = 0.9V _max requires a substrate content of »10K _m.

Figure 11. Binding interactions that determine the stereospecificity of an enzyme. They are illustrated for an enzyme that is specific for an (S)-enantiomer (with groups P ₁, P ₂, B, and X- bound to the asymmetric C atom) that has binding interactions with the S ₁, S ₂, and binding subsites. The correct binding of this enantiomer is shown to the left. The catalytic subsite (C) is then near the bond changed in the catalytic reaction. For the (R)-enantiomer, this and the other three binding interactions cannot occur simultaneously. Only two of the three possible binding interactions are possible. In the case shown to the right, one of these cases leads to a large distance between the catalytic site and the bond to be changed. Thus, either the binding of the (R)-enantiomer is weaker than that for the (S)-enantiomer or the catalytic reaction for the (R)-enantiomer is slower as the activation energy will increase (see Figure 8).

Figure 12. Different enzyme and chromatographic separation processes for the production of pure (S)-enantiomers from racemic mixtures by kinetic resolution or asymmetric synthesis from prochiral compounds. The class of enzymes that can catalyze the different reactions is given based on the classification given in Section 2. The enzyme processes I–IV are equilibrium controlled, with the exception of process IV, which is catalyzed by hydrolases in aqueous systems that are kinetically controlled reactions. In process I, a 100% product yield can be obtained, whereas in the other processes (II–V) only 50% of the racemic mixture can be converted to the desired enantiomer. To obtain 100% yield, the unwanted enantiomer must be racemized. A similar scheme can be designed for the production of the (R)-enantiomer.

Figure 13. Range of published stereoselectivities E _eq (Eq. ( 15)) for the S ₁ binding subsite (acyl binding in Figure 3) and E _kin (Eq. ( 16)) for the binding subsite (leaving group in equilibrium-controlled hydrolysis or nucleophile binding subsite in kinetically controlled synthesis) for enzymes (mainly hydrolases). Note that penicillin amidase has different stereospecificities in both binding subsites. The same applies for lipases and esterases, where the binding subsites are the acid and alcohol binding sites.

Figure 14. pH dependence of v, k _cat, and K _m for α-chymotrypsin-catalyzed hydrolysis of an uncharged substrate (from Bender et al., 1964).

Figure 15. Temperature dependence of the activity of different enzymes. Amylase from pancreas (1), Bacillus subtilis (2), and Bacillus licheniformis (3); peptidase from pancreas (4) and B. subtilis (5)(from Godfrey, 1996).

Figure 16. Temperature dependence of the stereoselectivity E _eq for different binding subsites of α-chymotrypsin and penicillin amidase in aqueous solution, and lipase in organic solvent. Bovine α-chymotrypsin: (S ₁ selectivity, see Figure 3) hydrolysis of N-acetyl-(R,S)-phenylglycine methyl ester (closed circles) and (R,S)-phenylglycine methyl ester (open circles) at pH 7.5 (Galunsky, Ignatova, and Kasche, 1997). Penicillin amidase from E. coli: (S ₁ selectivity) hydrolysis of (R,S)-phenylglycine amide (open squares), (R,S)-hydroxyphenylglycine ester (filled squares), and N-acetyl-(R,S)-phenylglycine (gray squares, selectivity) at pH 7.5 (Kasche et al., 1996). Lipase from P. cepacia (triangles): (nucleophile binding site) esterification of racemic azirine with vinyl acetate in diethyl ether (Sakai et al., 1997). Note that stereospecificity of penicillin amidase differs in the S ₁ and subsites.

Figure 17. Interactions of enzymes with inhibitors (I _c is a competitive inhibitor), modulators M (activators or noncompetitive inhibitors), and substrates S.

Figure 18. Graphical determination of the type of inhibition from Eadie–Hofstee plots with and without a constant concentration inhibitor. The type of substrate inhibition (competitive or noncompetitive) cannot be determined from such curves.

Figure 19. Ionic strength dependence of k _cat and K _m of an enzyme-catalyzed reaction for different charges of the substrate and the active site |Z _E| = |Z _S| = 1.

Figure 20. Calculated K _app/K _ref from Eq. ( 40) and experimental values (K _app/[H ₂O]) of pH dependence of the association constants for the formation of esters (a), peptide antibiotics (b, d), and peptides (c).(a) Ester synthesis from a carboxylic acid (); reference states: the uncharged compounds. (b) Synthesis of penicillin G () from phenylacetic acid () and 6-APA (, ); reference states: uncharged product and neutral acid and positively charged 6-APA. (c) Synthesis of a dipeptide from an amino-protected () and carboxyl-protected () amino acid; reference states: the uncharged compounds. (d) Experimental data for the synthesis of penicillin G at 25 °C (I = 0.2 M). Single, double, and triple primes denote the dissociation constants for the acid, base, and condensation product, respectively; subscript 1 denotes the acid dissociation constant and subscript 2 denotes the base dissociation constant.

Figure 21. The enantiomeric excess ee _p of the product (dashed line) and ee _S for the remaining substrate (solid line) as a function of the extent of the reaction for kinetic racemate resolutions catalyzed by (S)- or (R)-specific enzymes with different E-values (the E-values for the (R)-specific enzymes are the inverse of the given E-values). The horizontal dotted line shows that a product yield of ³45% with ee ³0.95 requires a (S)-specific enzyme with E ³ 100.

Figure 23. Kinetically controlled synthesis of soluble N-acetyl-(S)-Tyr-(S)-Arg-NH ₂ (ATAA, circles), a precursor of the dipeptide (Tyr-Arg) kyotorphin, from N-acetyl-(S)-Tyr-OEt (ATEE) as a suspension and soluble (S)-Arg-NH ₂ with the peptidase α-chymotrypsin (CT). Conditions: enzyme content 10 µg ml ^–1, 25 °C, pH 9.0 (carbonate buffer, I = 0.2 M); the pH is kept constant during the reaction. Squares: the soluble hydrolysis product N-acetyl-(S)-Tyr. Closed symbols: starting concentrations are 800 mM (S)-Arg-NH ₂ and 750 mM (total content) ATEE as a suspension. Open symbols: starting concentrations are 400 mM (S)-Arg-NH ₂ and 400 mM (total content) ATEE as a suspension. The dissolved ATEE concentration in the aqueous system during the reaction was <1 mM (Kasche and Galunsky, 1995).

Figure 22. The concentration gradients at the phase boundaries in enzyme-catalyzed processes in aqueous suspensions involving insoluble substrates or products. The asterisk denotes the concentration at the phase boundary at equilibrium between the solid and aqueous phases.

Figure 24. Structures of the two most often used ionic liquids 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF ₄) or its hexafluorophosphate (BMIM-PF ₆).

Figure 25. Enzyme-catalyzed process in a two-phase system. The enzyme-catalyzed process can only occur in the aqueous phase or at the interphase. K _aq and K _org are the equilibrium constants in the aqueous and organic phases, respectively; p _A, p _B, and p _C (=c _org/c _aq) are partition coefficients; p _aq and p _org are the partition coefficients for the solvents.

Figure 26. The rate of an enzyme-catalyzed reaction with low (systems II and III in Table 8) and high (system I in Table 8) water content as a function of total water content. The total enzyme amount in the system is kept constant.

Figure 27. Free energy diagram for the reversible denaturation of an enzyme with the activation energies for denaturation () and renaturation (). The native structure is formed spontaneously at normal temperatures. At higher temperatures or extreme pH values, the equilibrium is shifted to the unfolded (denatured) enzyme. The denatured enzyme can be renatured by slowly changing the temperature or pH to the original value. A rapid change “freezes” the denatured state, due to the slow rate of refolding. The renaturation must also be carried out at low enzyme concentrations to avoid aggregation of the denatured enzymes. A comparison with Figure 7 shows that enzymes are stabilized when they bind substrates.

Figure 28. The pH stability of different penicillin amidase forms from E. coli (closed circles: PA _7.0; open circles: PA _6.7; subscripts indicate their isoelectric point) and A. faecalis (cross symbols) at 25 °C. The half-life time of the monomolecular denaturation was measured in buffer with ionic strength 1 M by activity determinations (Wiesemann, 1991; A. Rieks and V. Kasche, unpublished data).

Figure 29. The specific rate of an enzyme calculated from Eq. ( 8) and expressed in k _cat/K _m units as a function of the substrate content [S] in the living cell or its environment expressed in K _m units. It is an evolutionary advantage to use as much as possible of the synthesized enzyme for catalytic conversion of the substrate. This occurs when [S] is 1–10K _m (shaded area). At higher ratios, the specific rate is independent of [S]; this is unfavorable for metabolic regulation.