Nod" 3 Hod NHoa*'2 Hod NH2~
4.6 ELEMENTARY STEPS IN BIOCATALYTIC REACTIONS .1 Introduction
4.6.3 General Features of Enzymes
4.6.3.1 Specificity
4 w B O N D I N G A N D E L E M E N T A R Y S T E P S I N C A T A L Y S I S 175 merization reaction will occur, but also the now undesirable cracking reaction, which is a consecutive reaction. The low alkene partial pressure reduces the carbenium ion concentration. The catalyst now performs two functions: metal- catalyzed CH activation and protonation of alkenes. For this reason the hydro- isomerization reaction according to Fig. 4.62 is called a bifunctional reaction.
4.6 ELEMENTARY STEPS IN BIOCATALYTIC REACTIONS
176 4 m BONDING AND ELEMENTARY STEPS IN CATALYSIS TABLE 4.1
The six categories of enzymes and the type of reaction catalyzed by each
N a m e Reaction catalyzed
1. Oxidoreductase 2. Transferases
3. Hydrolases 4. Lyases
5. Isomerases 6. Ligases
oxidation-reduction reactions
transfer of chemical group from one substrate to another or from one part of substrate to another
hydrolysis reactions
elimination of groups from adjacent atoms or addition of groups to dou- ble bonds
rearrangements (isomerizations)
formation of bonds to groups with hydrolysis of ATP, etc.
that is transformed, and to the identity of the products when more than one possible species can be produced [62,63].
Consider, for example, the proteases pronase, trypsin, and thrombin. All are endopeptidases, meaning that they hydrolytically cleave internal peptide bonds in a polypeptide chain. Although the same chemical reaction is catalyzed in all three cases, the three enzymes exhibit very different specificities. Pronase is a bacterial protease whose function is to hydrolyze proteins in order to provide amino acids as nutrients for growth; hence, it is essentially non-specific, and hydrolyzes virtually all peptide bonds at reasonable rates. In contrast, trypsin is a component of the mammalian digestive system and is also the key enzyme involved in activating other proteolytic enzymes by hydrolysis of specific peptide bonds in inactive precursor molecules; trypsin will only hydrolyze internal peptide bonds in which the carbonyl-containing amino acid bears a positively-charged (basic) side chain, i.e., arginine or lysine. Finally, thrombin is a component of the blood-clotting cascade, and its substrate is a protease that is the next member of the cascade and which is activated by proteolysis; as a result, thrombin is absolutely specific for cleaving an arginine- glycine peptide bond.
These specificities are summarized in Fig. 4.63.
As an example of enzyme specificity in distinguishing between chemically identical portions of a substrate molecule, consider the enzyme alcohol de- hydrogenase. This enzyme converts ethanol to acetaldehyde in the liver:
CH3CH2OH + NAD § ~ CH3CHO + NADH
Superficially, this looks like a simple reaction, but in fact it is completely stereo- specific. The enzyme can and does distinguish between the two chemically
4 ~ B O N D I N G A N D E L E M E N T A R Y STEPS IN CATALYSIS
~.
-I 0 II ]~. 0 IINH----CH--C N H ~ C H ~ C - - Scissile
t
peptide bond
pronase: Rn-1 = Rn = anything (almost) trypsin: Rn-1 = Arg, Lys only thrombin: Rn-1 = Arg, Rn = Gly only
Fig. 4.63. Specificity of selected endopeptidases for peptide bond cleavage.
177
OH
I
I
!
C H 3
Fig. 4.64. The prochiral methylene hydrogen atoms of ethanol.
identical but prochiral methylene hydrogen atoms of ethanol, and only the H-atom labelled H~ in Fig. 4.64 is transferred to the NAD § coenzyme, with Hb remaining in the acetaldehyde product. This illustrates a general and potentially very useful point: enzymes, because they are polymers of chiral building blocks, are intrinsically chiral and will always distinguish between prochiral groups of a substrate molecule.
As an additional example of enzymatic specificity, consider the enzyme DNA polymerase, which synthesizes a polymer of the four basic nucleic acid units (adenosine, guanosine, thymosine, and cytosine) on a template strand of DNA.
This enzyme exhibits an extraordinarily high degree of fidelity in the copies it produces: typically <1 error per 106 nucleotides polymerized.
A final example of the kind of specificity exhibited by enzymes is provided by squalene oxidocyclase. As shown in Fig. 4.65, this enzyme catalyzes the con- version of the 30-carbon isoprene derivative squalene 2,3-epoxide, which con- tains only one asymmetric carbon atom, to the steroid lanosterol in a single step that forms four new carbon-carbon bonds and seven new chiral centres in 100%
yield and with no isomeric impurities.
The aspects of enzyme-catalyzed reactions cited above are so far beyond what is normally achieved in chemical reactions that there is a tendency to believe that unusual principles must be invoked to explain the results. This is absolutely untrue: just like any other kind of catalysts, enzymes do chemistry, not magic/The goal of this and the next section is to illustrate how enzymatic reactions are controlled by familiar chemical principles of structure and reactivity.
178 4 m BONDING AND ELEMENTARY STEPS IN CATALYSIS
l
2,3-Oxidosqualene
i
I
H
L a n o s t e r o l
Fig. 4.65. The conversion of squalene-2,3-epoxide to lanosterol by squalene oxidocyclase.
Enzymes are capable of the kind of selectivity and rate enhancements dis- cussed above because their active sites exhibit a number of distinctive features compared to the active sites employed by soluble transition metal complexes and solid state catalysts: multi-point contact with the substrate, which is very hard to engineer in a synthetic catalyst; the structural flexibility to undergo collective and rapid changes in structure to facilitate catalysis of a reaction; and a unique ability to combine apparently incompatible features in catalysis, such as simultaneous acid and base catalysis and hydrophobic/hydrophilic interactions [62,63]. These points are discussed in more detail in the following sections.
4.6.3.2 Regulation
The activity of many enzymes is regulated physiologically in one or more of several ways. Any or all of these can become important in the design of a process that attempts to take advantage of the many desirable features exhibited by enzymes as catalysts, discussed in the previous section. At the molecular level, regulation of enzyme activity occurs in at least three distinct ways: feedback inhibition, allosteric regulation, and covalent modification. A detailed dis- cussion of these phenomena is outside the scope of this text, but the key points of each are summarized very briefly below. Although some of these features are similar to those exhibited by organometallic and solid state catalysts, biocatalytic systems are generally more complex, in that interactions remote from the active site are important in dictating the activity of the catalyst. The reader will recog- nize many features in common with the organometallic and solid state catalysts discussed in previous chapters, but biocatalytic systems are unique in the way in which complex reaction systems are coupled.
(a) Feedback inhibition ~ As shown schematically in Fig. 4.66, often the product of the last enzyme will inhibit the first in a sequence of several biosynthetic enzymes [64]. This can be an important consideration in applied biocatalysis if
4 -- BONDING AND ELEMENTARY STEPS IN CATALYSIS Enzyme
inhibited by Z
A --lll ~>B > C
L.
>D
~ > Z End product)
I
179
Fig. 4.66. Schematic illustration of the fact that the last product in a pathway often inhibits the first enzyme in that pathway (feedback inhibition).
the aim is to accumulate high concentrations of the last product of the pathway!
Feedback inhibition in this sense is a phenomenon that is distinct from the related phenomenon of product inhibition, which is generally exhibited by all catalysts, including enzymes. In product inhibition, the product that has been formed in the reaction simply competes with the substrate for the active site. In the case of feedback inhibition, the inhibitor is usually a molecule that may not be a close structural analog of the substrate or product. In addition, the inhibitor usually binds at a site that is distinct from and often remote from the active site, and exerts its effect on the enzyme's activity via allosteric interactions.
The term allosteric interactions refers to the ability of a substance to affect the catalytic activity of an enzyme by binding at a remote site, thereby inducing subtle structural changes that are transmitted via the protein structure to the active site. Such substances are referred to as allosteric effectors, and can act as activators, to increase the activity of an enzyme under specified conditions, or as inhibitors, which decrease the activity of the enzyme.
(b) Covalent modification - - Many enzymes can be turned on or off by addition or removal of a small group, often a phosphate, or by cleavage of the peptide chain [65]. Covalent modification by phosphorylation is illustrated schematic- ally in Fig. 4.67, and is very common. For example, the enzymes that metabolize glycogen (a polymeric form of glucose used for short-term energy storage) are regulated by phosphorylation of a single serine residue in the protein. Reversible phosphorylation of the serine -CH2OH group is used to turn on glycogen
~):G- ..Phosphorylation, .= ;
OH ~
ADP ATP
"ONp,~O 0 / N o
Fig. 4.67. Covalent modification of an enzyme by phosphorylation, which often results in altered catalytic properties.
180 4 ~ B O N D I N G A N D E L E M E N T A R Y STEPS IN CATALYSIS
Hydrolysis of specific
Activation peptide bonds
site . . . . . j
Inactive precursor Active enzyme
Fig. 4.68. Schematic drawing illustrating the selective cleavage of a peptide bond in a protein, which can result in altered enzymatic properties.
phosphorylase and to turn off glycogen synthetase in an inverse manner, en- suring that both enzymes are never fully functional at the same time. This phenomenon is distinct from activation of organometallic or solid state catalysts by ligand or solvent desorption, because it generally involves a modification of the structure that is remote from the active site, which results in subtle structural changes that are transmitted to the active site, much as is the case with allosteric interactions.
Some e n z y m e s are synthesized as inactive precursors (zymogens) that are activated by cleavage of one or more peptide bonds, as shown schematically in Fig. 4.68. Probably the best-characterized examples are found among digestive enzymes such as chymotrypsin, which is synthesized as an inactive precursor that consists of a single polypeptide chain containing 245 amino acids. It is activated by proteolytic cleavage of the peptide chain in two places and removal of amino acids number 14, 15, 147 and 148.
Again, these processes are distinct from those typically observed in solid or organometallic complexes, where precursors are often activated by ligand dis- sociation or solvent desorption, in that the covalent modification occurs at a site that is remote from the active site.
4.6.3.3 Active sites
The fact that enzymes form stoichiometric enzyme-substrate (E.S) complexes (Chapter 3) suggests that substrates bind at a specific site on the enzyme, which presumably contains the functional groups that interact directly with the sub- strate during catalysis. Although the details of active structure vary greatly from one enzyme to another, the following general statements can reliably be made.
(a) Active sites are a small part of the overall enzyme molecule. Most enzymes consist of >100 amino acids and are roughly globular proteins that are >25/~ in diameter. In contrast, the active site of the enzyme is of roughly the same size as the substrate or, in the case of polymeric substrates or products such as glycogen or nucleic acids, the monomeric unit of the polymer.
4 - - BONDING AND ELEMENTARY STEPS IN CATALYSIS 181
(b) Active sites are larger than the substrate. The enzyme at least partially surrounds the substrate during catalysis, providing a sheltered microenviron- ment that contains the right combination of functional groups in the proper location to effect catalysis.
(c) Active sites tend to be clefts or crevices in an enzyme, lined with appropriate amino acid side chains. Polar groups are used for both acid-base catalysis and binding, and non-polar groups are used for hydrophobic binding. Water is generally excluded from the active site unless it is a reactant, and catalysis therefore occurs in a non-aqueous local environment. The amino acid side chains at the active site usually come from residues that are widely separated in the amino acid sequence (e.g., lysozyme uses amino acids # 35, 52, 62, 63, 101 at its active site). The complex three-dimensional structure of the protein results in the juxtaposition of these groups.
(d) Substrate binding to the active site tends to be relatively weak. The equilibrium constant for formation of the E.S or E.P complex is typically only ca. 10 -2 to 10 -s M, corresponding to a value of AG = -12 t o - 5 0 kJ/mol. This is an important point because an enzyme must be able to release its product readily in order to turn over rapidly. If the E.S complex is too stable, then the depth of the valleys in the energy vs. reaction coordinate profile can become so great that the activation energy is actually greater than for the uncatalyzed reaction. Thus, excessively high affinity of the enzyme for the substrate by itself is counterproductive, and would lead to an anti-catalyst!
(e) The specificity of an enzyme depends on complementary structures of substrate and active site. This is the so-called lock and key model, shown schematically on the left in Fig. 4.69. Although the drawing shows only complementarity in shape between the enzyme and the substrate, it is important to note that comple- mentarity in both shape and charge is usually observed. In fact, the lock and key model is oversimplified: most enzymes have less rigid active sites that fold around the substrate during formation of the E.S complex. This is the induced-fit model, shown schematically on the right in Fig. 4.69 for comparison.
S u bst rate
(
S u bstrate+ .. , ) + >
! \ \ /
a b ,/ ES complex ES complex
~ Lock and Key Induced-fit
Fig. 4.69. C o m p a r i s o n of the lock a n d k e y vs. the i n d u c e d - f i t m o d e l s for e n z y m e - s u b s t r a t e interaction.
182 4 - - B O N D I N G A N D E L E M E N T A R Y STEPS IN CATALYSIS
In summary, enzyme active sites exhibit a number of distinctive features that differ from those of the active sites employed by soluble transition metal complexes and solid state catalysts: multi-point contact with the substrate, which is very hard to engineer in a synthetic catalyst; the structural flexibility to undergo collective and rapid changes in structure to facilitate catalysis of a reaction; and a unique ability to combine apparently incompatible features in catalysis, such as simultaneous acid and base catalysis and hydrophobic/
hydrophilic interactions.
4.6.4 Factors Important In Enzymatic Catalysis