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Chapter 7: Enzymes: Mechanism of Action
Peter J. Kennelly; Victor W. Rodwell
OBJECTIVES
OBJECTIVES
After studying this chapter, you should be able to:
Describe the structural relationships between specific B vitamins and certain coenzymes.
Outline the four principal mechanistic strategies employed by enzymes to catalyze chemical reactions.
Explain the concepts of the “lock and key” and “induced fit” models for enzyme–substrate interaction and how the latter accounts for the
dynamic nature of enzyme catalysis.
Outline the underlying principles of enzyme­linked immunoassays.
Describe how detecting and measuring the activity of many enzymes can be facilitated by coupling with an appropriate dehydrogenase.
Identify proteins whose plasma levels are used as biomarkers for diagnosis and prognosis.
Describe the application of restriction endonucleases and of restriction fragment length polymorphisms in the detection of genetic diseases.
Illustrate the utility of site­directed mutagenesis for the identification of aminoacyl residues that are involved in the recognition of substrates or
allosteric effectors, or in the mechanism of catalysis.
Describe how “affinity tags” can facilitate purification of a protein expressed from its cloned gene.
Discuss the events that led to the discovery that RNAs can act as enzymes, and briefly describe the evolutionary concept of an “RNA world.”
BIOMEDICAL IMPORTANCE
Nobel laureate (1946) James Sumner defined an enzyme as “a catalyst of high molecular weight and biological origin.” Enzymes catalyze the chemical
reactions that make life on the earth possible, such as the breakdown of nutrients to supply energy and biomolecular building blocks; the assembly of
those building blocks into proteins, DNA, membranes, cells, and tissues; and the harnessing of energy to power cell motility, neural function, and
muscle contraction. Almost all enzymes are proteins. Notable exceptions include ribosomal RNAs and a handful of RNA molecules imbued with
endonuclease or nucleotide ligase activity known collectively as ribozymes. Many pathologic conditions are the direct consequence of changes in the
quantity or in the catalytic activity of key enzymes that result from genetic defects, nutritional deficits, tissue damage, toxins, or infection by viral or
bacterial pathogens (eg, Vibrio cholerae). Thus, the ability to detect and to quantify the activity of specific enzymes in blood, other tissue fluids, or cell
extracts provides information that enhances the physician’s ability to diagnose many diseases.
In addition to serving as the catalysts for all metabolic processes, the impressive catalytic activity and substrate specificity of enzymes enables them to
fulfill unique roles in human health and well­being. The protease rennin, for example, is utilized in the production of cheeses, while lactase is
employed to remove lactose from milk to benefit lactose­intolerant individuals. Proteases and amylases augment the capacity of detergents to remove
dirt and stains, while other enzymes can participate in the stereospecific synthesis of complex drugs or antibiotics.
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Chapter
7: Enzymes:
of Action,
Peter J. Kennelly;
Victor W. Rodwell
ENZYMES
AREMechanism
EFFICIENT
& HIGHLY
SPECIFIC
CATALYSTS
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Page 1 / 19
The enzymes that catalyze the conversion of one or more compounds (substrates) into one or more different compounds (products) generally
extracts provides information that enhances the physician’s ability to diagnose many diseases.
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In addition to serving as the catalysts for all metabolic processes, the impressive catalytic activity and substrate
specificity
of enzymes enables them to
fulfill unique roles in human health and well­being. The protease rennin, for example, is utilized in the production of cheeses, while lactase is
employed to remove lactose from milk to benefit lactose­intolerant individuals. Proteases and amylases augment the capacity of detergents to remove
dirt and stains, while other enzymes can participate in the stereospecific synthesis of complex drugs or antibiotics.
ENZYMES ARE EFFICIENT & HIGHLY SPECIFIC CATALYSTS
The enzymes that catalyze the conversion of one or more compounds (substrates) into one or more different compounds (products) generally
enhance the rates of the corresponding noncatalyzed reaction by factors of 106 to 109 or even more. Although some enzymes become modified during
catalysis, these transient alterations are resolved in the course of the reaction being catalyzed. In addition to being highly efficient, enzymes are also
extremely selective. Typically, the catalysts used in synthetic chemistry are specific for the type of reaction catalyzed and will act on any compound
containing the relevant functional group. Enzymes, however, are generally specific for a single substrate or even a single stereoisomer thereof—for
example, D­ but not L­sugars, L­ but not D­amino acids—or a small set of closely related substrates. Since they bind substrates through at least “three
points of attachment” (Figure 7–1), enzymes also can produce chiral products from nonchiral substrates. For example, reduction of the nonchiral
substrate pyruvate by lactate dehydrogenase produces exclusively L­lactate, not a racemic mixture of D­ and L­lactate. The exquisite specificity of
enzyme catalysts imbues living cells with the ability to simultaneously conduct and independently control a broad spectrum of biochemical processes.
FIGURE 7–1
Planar representation of the “three­point attachment” of a substrate to the active site of an enzyme. Although atoms 1 and 4 are
identical, once atoms 2 and 3 are bound to their complementary sites on the enzyme, only atom 1 can bind. Once bound to an enzyme, apparently
identical atoms thus may be distinguishable, permitting a stereospecific chemical change.
ENZYMES ARE CLASSIFIED BY REACTION TYPE
Some of the names for enzymes first described in the earliest days of biochemistry persist in use to this day. Examples include pepsin, trypsin, and
amylase. Early biochemists generally named newly discovered enzymes by adding the suffix –ase to a descriptor for the type of reaction catalyzed. For
example, enzymes that remove the elements of hydrogen, H2 or H− plus H+, generally are referred to as dehydrogenases, enzymes that hydrolyze
proteins as proteases, and enzymes that catalyze rearrangements in configuration as isomerases. In many cases, these general descriptors were
supplemented with terms indicating the particular substrate on which the enzyme acts (xanthine oxidase), its source (pancreatic ribonuclease), its
mode of regulation (hormone­sensitive lipase), or a characteristic feature of its mechanism of action (cysteine protease). Where needed, alphanumeric
designators can be added to identify multiple forms, or isozymes, of an enzyme (eg, RNA polymerase III; protein kinase Cβ).
As more enzymes were discovered, these early naming conventions increasingly resulted in the inadvertent designation of some enzymes by multiple
names or the assignment of duplicate names to enzymes exhibiting similar catalytic capabilities. To address these problems, the International Union of
Biochemistry (IUB) developed a system of enzyme nomenclature in which each enzyme has a unique name and code number that identify the type of
reaction catalyzed and the substrates involved. Enzymes are grouped into the following six classes:
1. Oxidoreductases—enzymes that catalyze oxidations and reductions
2. Transferases—enzymes that catalyze transfer of moieties such as glycosyl, methyl, or phosphoryl groups
3. Hydrolases—enzymes
that
cleavage of C—C, C—O, C—N, and other covalent bonds
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Chapter 7: Enzymes: Mechanism of Action, Peter J. Kennelly; Victor W. Rodwell
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4. Lyases—enzymes
that
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andPolicy
other •covalent
by atom elimination, generating double bonds
5. Isomerases—enzymes that catalyze geometric or structural changes within a molecule
reaction catalyzed and the substrates involved. Enzymes are grouped into the following six classes:
1. Oxidoreductases—enzymes that catalyze oxidations and reductions
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2. Transferases—enzymes that catalyze transfer of moieties such as glycosyl, methyl, or phosphoryl groups
3. Hydrolases—enzymes that catalyze hydrolytic cleavage of C—C, C—O, C—N, and other covalent bonds
4. Lyases—enzymes that catalyze cleavage of C—C, C—O, C—N, and other covalent bonds by atom elimination, generating double bonds
5. Isomerases—enzymes that catalyze geometric or structural changes within a molecule
6. Ligases—enzymes that catalyze the joining together (ligation) of two molecules in reactions coupled to the hydrolysis of ATP
The IUB name of hexokinase is ATP:D­hexose 6­phosphotransferase E.C. 2.7.1.1. This name identifies hexokinase as a member of class 2 (transferases),
subclass 7 (transfer of a phosphoryl group), sub­subclass 1 (alcohol is the phosphoryl acceptor), and “hexose­6” indicates that the alcohol
phosphorylated is on carbon six of a hexose. While EC numbers have proven particularly useful to differentiate enzymes with similar functions or
similar catalytic activities, IUB names tend to be lengthy and cumbersome. Consequently, hexokinase and many other enzymes commonly are referred
to using their traditional, albeit sometimes ambiguous names.
PROSTHETIC GROUPS, COFACTORS, & COENZYMES PLAY IMPORTANT ROLES IN CATALYSIS
Many enzymes contain small molecules or metal ions that participate directly in substrate binding or in catalysis. Termed prosthetic groups,
cofactors, and coenzymes, they extend the repertoire of mechanistic capabilities beyond those afforded by the functional groups present on the
aminoacyl side chains of peptides.
Prosthetic Groups
Prosthetic groups are tightly and stably incorporated into a protein’s structure by covalent bonds or noncovalent forces. Examples include pyridoxal
phosphate, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), thiamin pyrophosphate, lipoic acid, biotin, and transition metals such as
Fe, Co, Cu, Mg, Mn, and Zn. Metal ions that participate in redox reactions generally are bound as organometallic complexes such as the prosthetic
groups heme or iron–sulfur clusters (see Chapter 10). Metals may facilitate the binding and orientation of substrates, the formation of covalent bonds
with reaction intermediates (Co2+ in coenzyme B12, see Chapter 44), or by acting as Lewis acids or bases to render substrates more electrophilic
(electron­poor) or nucleophilic (electron­rich), and hence more reactive (see Chapter 10).
Cofactors Associate Reversibly With Enzymes or Substrates
Cofactors serve functions similar to those of prosthetic groups and overlap with them. The major difference between the two is operational, not
chemical. Cofactors bind weakly and transiently to their cognate enzymes or substrates, forming dissociable complexes. Therefore, unlike associated
prosthetic groups, cofactors must be present in the surrounding environment to promote complex formation in order for catalysis to occur. Metal ions
form the most numerous class of cofactors. Enzymes that require a metal ion cofactor are termed metal­activated enzymes to distinguish them
from the metalloenzymes for which bound metal ions serve as prosthetic groups. It is estimated that one­third of all enzymes fall into one of these
two groups.
Many Coenzymes, Cofactors, & Prosthetic Groups Are Derivatives of B Vitamins
The water­soluble B vitamins supply important components of numerous coenzymes. Nicotinamide is a component of the redox coenzymes NAD and
NADP (Figure 7–2); riboflavin is a component of the redox coenzymes FMN and FAD; pantothenic acid is a component of the acyl group carrier
coenzyme A. As its pyrophosphate thiamin participates in the decarboxylation of α­keto acids while folic acid and cobamide coenzymes function in
one­carbon metabolism. In addition, several coenzymes contain the adenine, ribose, and phosphoryl moieties of AMP or ADP (see Figure 7–2).
FIGURE 7–2
Structure of NAD+ and NADP+ . For NAD+, OR = —OH. For NADP+, —OR = —OPO32–.
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FIGURE 7–2
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Structure of NAD+ and NADP+ . For NAD+, OR = —OH. For NADP+, —OR = —OPO32–.
Coenzymes Serve as Substrate Shuttles
Coenzymes serve as recyclable shuttles that transport many substrates from one point within the cell to another. The function of these shuttles is
twofold. First, they stabilize species such as hydrogen atoms (FADH2) or hydride ions (NADH) that are too reactive to persist for any significant time in
the presence of the water, oxygen, or the organic molecules that permeate cells. Second, they increase the number of points of contact between
substrate and enzyme, which increases the affinity and specificity with which small chemical groups such as acetate (coenzyme A), glucose (UDP), or
hydride (NAD+) are bound by their target enzymes. Other chemical moieties transported by coenzymes include methyl groups (folates) and
oligosaccharides (dolichol).
CATALYSIS OCCURS AT THE ACTIVE SITE
An important early 20th­century insight into enzymic catalysis sprang from the observation that the presence of substrates renders enzymes more
resistant to the denaturing effects of elevated temperatures. This observation led Emil Fischer to propose that enzymes (E) and their substrates (S)
form an enzyme–substrate (ES) complex whose thermal stability is greater than that of the enzyme itself. This insight profoundly shaped our
understanding of both the chemical nature and kinetic behavior of enzymic catalysis.
Fischer reasoned that the exquisitely high specificity with which enzymes discriminate their substrates when forming an ES complex was analogous to
the manner in which a mechanical lock distinguishes the proper key. The analogy to enzymes is that the “lock” is formed by a surface­accessible cleft
or pocket in the enzyme called the active site (see Figures 5–6 and 5–8). As implied by the adjective “active,” the active site is much more than simply a
recognition site for binding substrates; it provides the environment wherein chemical transformation takes place. Within the active site, substrates are
brought into close proximity with one another in optimal alignment with the cofactors, prosthetic groups, and aminoacyl side chains that participate in
catalyzing the transformation of substrates into products (Figure 7–3). Catalysis is further enhanced by the capacity of the active site to shield
substrates from water and generate an environment whose polarity, hydrophobicity, acidity, or alkalinity can differ markedly from that of the
surrounding cytoplasm.
FIGURE 7–3
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Two­dimensional
representation
substrate, glycyl­tyrosine, bound within the active site of carboxypeptidase A.
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Chapter 7: Enzymes: Mechanism of Action, Peter J. Kennelly; Victor W. Rodwell
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catalyzing the transformation of substrates into products (Figure 7–3). Catalysis is further enhanced by the capacity of the active site to shield
substrates from water and generate an environment whose polarity, hydrophobicity, acidity, or alkalinityAteneo
can differ
markedly
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FIGURE 7–3
Two­dimensional representation of a dipeptide substrate, glycyl­tyrosine, bound within the active site of carboxypeptidase A.
ENZYMES EMPLOY MULTIPLE MECHANISTIC STRATEGIES TO FACILITATE CATALYSIS
Enzymes use combinations of four general mechanistic strategies to achieve dramatic enhancements of the rates of chemical reactions.
Catalysis by Proximity
In order to chemically interact, substrate molecules must come within bond­forming distance of one another. The higher their concentration, the more
frequently they will encounter one another, and the greater will be the rate at which reaction products appear. When an enzyme binds substrate
molecules at its active site, it creates a region of high local substrate concentration, one in which they are oriented in an ideal position to chemically
interact. In and of itself, this proximity results in rate enhancements of at least a 1000­fold over that observed in the absence of an enzyme.
Acid–Base Catalysis
In addition to contributing to the ability of the active site to bind substrates, the ionizable functional groups of aminoacyl side chains and, where
present, of prosthetic groups, can contribute to catalysis by acting as acids or bases. We distinguish two types of acid–base catalysis. Specific acid or
base catalysis refers to reactions for which the only participating acids or bases are protons or hydroxide ions. The rate of reaction thus is sensitive
to changes in the concentration of protons or hydroxide ions, but is independent of the concentrations of other acids (proton donors) or bases
(proton acceptors) present in the solution or at the active site. Reactions whose rates are responsive to all the acids or bases present are said to be
subject to general acid catalysis or general base catalysis.
Catalysis by Strain
For catalysis of lytic reactions, which involve breaking a covalent bond, enzymes typically bind their substrates in a conformation that weakens the
bond targeted for cleavage through physical distortion and electronic polarization. This strained conformation mimics that of the transition state
intermediate, a transient species that represents the midway point in the transformation of substrates to products. Nobel Laureate Linus Pauling was
the first to suggest a role for transition state stabilization as a general mechanism by which enzymes accelerate the rates of chemical reactions.
Knowledge of the transition state of an enzyme­catalyzed reaction is frequently exploited by chemists to design and synthesize more effective enzyme
inhibitors, called transition state analogs, as potential pharmacophores.
Covalent Catalysis
The process of2023­9­2
covalent
catalysis
involves
the formation of a covalent bond between the enzyme and one or more substrates. The modified
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Chapter
Enzymes:
Mechanism
of Action,
Peter
J. Kennelly;
Victor
W.reaction
Rodwellpathway whose activation energy is lower—and rate of reaction
enzyme 7:
thus
becomes
a reactant.
Covalent
catalysis
provides
a new
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therefore faster—than the pathways available in homogeneous solution. The chemically modified state of the enzyme is, however, transient.
Completion of the reaction returns the enzyme to its original, unmodified state. Its role thus remains catalytic. Covalent catalysis is particularly
common among enzymes that catalyze group transfer reactions. Residues on the enzyme that participate in covalent catalysis generally are
the first to suggest a role for transition state stabilization as a general mechanism by which enzymes accelerate the rates of chemical reactions.
Knowledge of the transition state of an enzyme­catalyzed reaction is frequently exploited by chemists to Ateneo
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Covalent Catalysis
The process of covalent catalysis involves the formation of a covalent bond between the enzyme and one or more substrates. The modified
enzyme thus becomes a reactant. Covalent catalysis provides a new reaction pathway whose activation energy is lower—and rate of reaction
therefore faster—than the pathways available in homogeneous solution. The chemically modified state of the enzyme is, however, transient.
Completion of the reaction returns the enzyme to its original, unmodified state. Its role thus remains catalytic. Covalent catalysis is particularly
common among enzymes that catalyze group transfer reactions. Residues on the enzyme that participate in covalent catalysis generally are
cysteine or serine, and occasionally histidine. Covalent catalysis often follows a “ping­pong” mechanism—one in which the first substrate is bound and
its product released prior to the binding of the second substrate (Figure 7–4).
FIGURE 7–4
“Ping­pong” mechanism for transamination. E—CHO and E—CH2NH2 represent the enzyme­pyridoxal phosphate and enzyme­pyridoxamine
complexes, respectively. (Ala, alanine; Glu, glutamate; KG, α­ketoglutarate; Pyr, pyruvate.)
SUBSTRATES INDUCE CONFORMATIONAL CHANGES IN ENZYMES
While Fischer’s “lock and key model” accounted for the exquisite specificity of enzyme–substrate interactions, the implied rigidity of the enzyme’s
active site failed to account for the dynamic changes that accompany catalytic transformations. This drawback was addressed by Daniel Koshland’s
induced fit model, which states that as substrates bind to an enzyme, they induce a conformational change that is analogous to placing a hand
(substrate) into a glove (enzyme) (Figure 7–5). The enzyme in turn induces reciprocal changes in its substrates, harnessing the energy of binding to
facilitate the transformation of substrates into products. The induced fit model has been amply confirmed by biophysical studies of enzyme motion
during substrate binding.
FIGURE 7–5
Two­dimensional representation of Koshland’s induced fit model of the active site of a lyase. Binding of the substrate A—B induces
conformational changes in the enzyme that align catalytic residues which participate in catalysis and strain the bond between A and B, facilitating its
cleavage.
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Chapter 7: Enzymes: Mechanism of Action, Peter J. Kennelly; Victor W. Rodwell
HIV
ILLUSTRATES
ACID–BASE
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Enzymes of the aspartic protease family, which includes the digestive enzyme pepsin, the lysosomal cathepsins, and the protease produced by the
FIGURE 7–5
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Two­dimensional representation of Koshland’s induced fit model of the active site of a lyase. Binding of the substrate A—B induces
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conformational changes in the enzyme that align catalytic residues which participate in catalysis and strain the bond between A and B, facilitating its
cleavage.
HIV PROTEASE ILLUSTRATES ACID–BASE CATALYSIS
Enzymes of the aspartic protease family, which includes the digestive enzyme pepsin, the lysosomal cathepsins, and the protease produced by the
human immunodeficiency virus (HIV), share a common mechanism that employs two conserved aspartyl residues as acid–base catalysts. In the first
stage of the reaction, one aspartate functions as a general base (Asp X, Figure 7–6) that extracts a proton from a water molecule to make it more
nucleophilic. The resulting nucleophile then attacks the electrophilic carbonyl carbon of the peptide bond targeted for hydrolysis, forming a
tetrahedral transition state intermediate. A second aspartate (Asp Y, Figure 7–6) then facilitates the decomposition of this tetrahedral
intermediate by donating a proton to the amino group produced by rupture of the peptide bond. The two active site aspartates can act simultaneously
as a general base or as a general acid because their immediate environment favors ionization of one, but not the other.
FIGURE 7–6
Mechanism for catalysis by an aspartic protease such as HIV protease. Curved arrows indicate directions of electron movement. ➀ Aspartate
X acts as a base to activate a water molecule by abstracting a proton. ➁ The activated water molecule attacks the peptide bond, forming a transient
tetrahedral intermediate. ➂ Aspartate Y acts as an acid to facilitate breakdown of the tetrahedral intermediate and release of the split products by
donating a proton to the newly formed amino group. Subsequent shuttling of the proton on Asp X to Asp Y restores the protease to its initial state.
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Mechanism for catalysis by an aspartic protease such as HIV protease. Curved arrows indicate directions of electron movement. ➀ Aspartate
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X acts as a base to activate a water molecule by abstracting a proton. ➁ The activated water molecule attacks the peptide bond, forming a transient
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tetrahedral intermediate. ➂ Aspartate Y acts as an acid to facilitate breakdown of the tetrahedral intermediate and release of the split products by
donating a proton to the newly formed amino group. Subsequent shuttling of the proton on Asp X to Asp Y restores the protease to its initial state.
CHYMOTRYPSIN & FRUCTOSE­2, 6­BISPHOSPHATASE ILLUSTRATE COVALENT CATALYSIS
Chymotrypsin
While catalysis by aspartic proteases involves the direct hydrolytic attack of water on a peptide bond, catalysis by the serine protease chymotrypsin
involves formation of a covalent acyl­enzyme intermediate. A conserved seryl residue, serine 195 in the bovine form, is activated via interactions with
histidine 57 and aspartate 102. While these three residues are far apart in primary structure, in the active site of the mature, folded protein they reside
within bond­forming distance of one another. Aligned in the order Asp 102­His 57­Ser 195, this trio forms a linked charge­relay network that acts as
a “proton shuttle.”
Binding of substrate initiates proton shifts that in effect transfer the hydroxyl proton of Ser 195 to Asp 102 (Figure 7–7). The enhanced nucleophilicity
of the seryl oxygen facilitates its attack on the carbonyl carbon of the peptide bond of the substrate, forming a covalent acyl­enzyme intermediate.
The proton on Asp 102 then shuttles via His 57 to the amino group liberated when the peptide bond is cleaved. The portion of the original peptide with
a free amino group then leaves the active site and is replaced by a water molecule. The charge­relay network now activates the water molecule by
withdrawing a proton through His 57 to Asp 102. The resulting hydroxide ion attacks the acyl­enzyme intermediate, and a reverse proton shuttle
returns a proton to Ser 195, restoring its original state. While modified during the process of catalysis, chymotrypsin emerges unchanged on
completion of the reaction. The proteases trypsin and elastase employ a similar catalytic mechanism, but the numbering of the residues in their Ser­
His­Asp proton
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Catalysis by chymotrypsin. ➀ The charge­relay system removes a proton from Ser 195, making it a stronger nucleophile. ➁ Activated Ser 195
The proton on Asp 102 then shuttles via His 57 to the amino group liberated when the peptide bond is cleaved. The portion of the original peptide with
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a free amino group then leaves the active site and is replaced by a water molecule. The charge­relay network
nowSchool
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withdrawing a proton through His 57 to Asp 102. The resulting hydroxide ion attacks the acyl­enzyme intermediate,
and a reverse proton shuttle
returns a proton to Ser 195, restoring its original state. While modified during the process of catalysis, chymotrypsin emerges unchanged on
completion of the reaction. The proteases trypsin and elastase employ a similar catalytic mechanism, but the numbering of the residues in their Ser­
His­Asp proton shuttles differ.
FIGURE 7–7
Catalysis by chymotrypsin. ➀ The charge­relay system removes a proton from Ser 195, making it a stronger nucleophile. ➁ Activated Ser 195
attacks the peptide bond, forming a transient tetrahedral intermediate. ➂ Release of the amino terminal peptide is facilitated by donation of a proton
to the newly formed amino group by His 57 of the charge­relay system, yielding an acyl­Ser 195 intermediate. ➃ His 57 and Asp 102 collaborate to
activate a water molecule, which attacks the acyl­Ser 195, forming a second tetrahedral intermediate. ➄ The charge­relay system donates a proton to
Ser 195, facilitating breakdown of the tetrahedral intermediate to release the carboxyl terminal peptide ➅.
Fructose­2,6­Bisphosphatase
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Chapter
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of Action,
Peter J.
Victor W.
Rodwell
Fructose­2,6­bisphosphatase,
a regulatory
enzyme
ofKennelly;
gluconeogenesis
(see
Chapter 19), catalyzes the hydrolytic release of the phosphate on
carbon
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of fructose­2,6­bisphosphate. Figure 7–8 illustrates the roles of seven active site residues. Catalysis involves a “catalytic triad” consisting of one Glu
and two His residues, of which one His forms a covalent phosphohistidyl intermediate.
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Fructose­2,6­Bisphosphatase
Fructose­2,6­bisphosphatase, a regulatory enzyme of gluconeogenesis (see Chapter 19), catalyzes the hydrolytic release of the phosphate on carbon 2
of fructose­2,6­bisphosphate. Figure 7–8 illustrates the roles of seven active site residues. Catalysis involves a “catalytic triad” consisting of one Glu
and two His residues, of which one His forms a covalent phosphohistidyl intermediate.
FIGURE 7–8
Catalysis by fructose­2,6­bisphosphatase. (1) Lys 356 and Arg 257, 307, and 352 stabilize the quadruple negative charge of the substrate by
charge–charge interactions. Glu 327 stabilizes the positive charge on His 392. (2) The nucleophile His 392 attacks the C­2 phosphoryl group and
transfers it to His 258, forming a phosphoryl­enzyme intermediate. Fructose­6­phosphate now leaves the enzyme. (3) Nucleophilic attack by a water
molecule, possibly assisted by Glu 327 acting as a base, forms inorganic phosphate. (4) Inorganic orthophosphate is released from Arg 257 and Arg
307. (Reproduced with permission from Pilkis SJ, Claus TH, Kurland IJ, et al: 6­Phosphofructo­2­kinase/fructose­2,6­bisphosphatase: a metabolic
signaling enzyme, Annu Rev Biochem. 1995;64:799­835.)
CATALYTIC RESIDUES ARE HIGHLY CONSERVED
Members of an enzyme family such as the aspartic or serine proteases employ a similar mechanism to catalyze a common reaction type, but act on
different substrates. Most enzyme families appear to have arisen through gene duplication events that created a second copy of the gene that encodes
a particular enzyme. The two genes, and consequently their encoded proteins, can then evolve independently, forming divergent homologs that
recognize different substrates. The result is illustrated by chymotrypsin, which cleaves peptide bonds on the carboxyl terminal side of large
hydrophobic amino acids, and trypsin, which cleaves peptide bonds on the carboxyl terminal side of basic amino acids. Proteins that diverged from a
common ancestor are said to be homologous to one another. The common ancestry of enzymes can be inferred from the presence of specific amino
acids in the same relative position in each family member. These residues are said to be evolutionarily conserved.
ISOZYMES ARE DISTINCT ENZYME FORMS THAT CATALYZE THE SAME REACTION
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catalysts
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ariseReserved.
through gene
duplication
in higher
eukaryotes,
mRNA splicing (see Chapter 36). While the homologous
proteases described act on different substrates, isozymes also may differ in auxiliary features such as sensitivity to particular regulatory factors (see
Chapter 9) or subcellular location that adapt them to specific tissues or circumstances rather than distinct substrates. Isozymes that catalyze the
recognize different substrates. The result is illustrated by chymotrypsin, which cleaves peptide bonds on the carboxyl terminal side of large
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hydrophobic amino acids, and trypsin, which cleaves peptide bonds on the carboxyl terminal side of basic amino acids. Proteins that diverged from a
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common ancestor are said to be homologous to one another. The common ancestry of enzymes can be inferred from the presence of specific amino
acids in the same relative position in each family member. These residues are said to be evolutionarily conserved.
ISOZYMES ARE DISTINCT ENZYME FORMS THAT CATALYZE THE SAME REACTION
Higher organisms often elaborate several physically distinct versions of a given enzyme, each of which catalyzes the same reaction. These protein
catalysts or isozymes can arise through gene duplication or, in higher eukaryotes, alternative mRNA splicing (see Chapter 36). While the homologous
proteases described act on different substrates, isozymes also may differ in auxiliary features such as sensitivity to particular regulatory factors (see
Chapter 9) or subcellular location that adapt them to specific tissues or circumstances rather than distinct substrates. Isozymes that catalyze the
identical reaction may also enhance survival by providing a “backup” copy of an essential enzyme.
THE CATALYTIC ACTIVITY OF ENZYMES FACILITATES THEIR DETECTION
The relatively small quantities of enzymes typically contained in cells hamper determination of their presence and abundance. However, the ability to
rapidly transform thousands of molecules of a specific substrate into products imbues each enzyme with the ability to amplify its presence. Under
appropriate conditions (see Chapter 8), the rate of the catalytic reaction being monitored is proportionate to the amount of enzyme present, which
allows its concentration to be inferred. Assays of the catalytic activity of enzymes are frequently used both in research and clinical laboratories.
Single­Molecule Enzymology
The limited sensitivity of traditional enzyme assays necessitates the use of a large group, or ensemble, of enzyme molecules in order to produce
measurable quantities of product. The data obtained thus reflect the average activity of individual enzymes across multiple cycles of catalysis. Recent
advances in nanotechnology and imaging have made it possible to observe catalytic events involving discrete enzyme and substrate molecules.
Consequently, scientists can now measure the rate of individual catalytic events, and sometimes a specific step in catalysis, by a process called single­
molecule enzymology, an example of which is illustrated in Figure 7–9.
FIGURE 7–9
Direct observation of single DNA cleavage events catalyzed by a restriction endonuclease. DNA molecules immobilized to beads (blue) are
placed in a flowing stream of buffer (black arrows), which causes them to assume an extended conformation. Cleavage at one of the restriction sites
(orange) by an endonuclease leads to a shortening of the DNA molecule, which can be observed directly in a microscope since the nucleotide bases in
DNA are fluorescent. Although the endonuclease (red) does not fluoresce, and hence is invisible, the progressive manner in which the DNA molecule is
shortened (1→4) reveals that the endonuclease binds to the free end of the DNA molecule and moves along it from site to site.
Drug Discovery Requires Enzyme Assays Suitable for High­Throughput Screening
Enzymes are frequent targets for the development of drugs and other therapeutic agents. These generally take the form of enzyme inhibitors (see
Chapter 8). The discovery of new drugs is greatly facilitated when a large number of potential pharmacophores can be simultaneously assayed in a
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rapid,
automated
fashion—a
process
referredPeter
to asJ.
high­throughput
screening
and 11 / 19
Chapter
7: Enzymes:
Mechanism
of Action,
Kennelly; Victor W.
Rodwell (HTS). HTS employs robotics, optics, data processing,Page
microfluidics
to simultaneously
conduct andTerms
monitor
of parallel
It thus serves as a perfect complement to combinatorial
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Hill. All Rights Reserved.
of thousands
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Notice assays.
• Accessibility
chemistry, a method for generating large libraries of chemical compounds spanning all possible combinations of a given set of chemical precursors.
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Drug Discovery Requires Enzyme Assays Suitable for High­Throughput Screening
Enzymes are frequent targets for the development of drugs and other therapeutic agents. These generally take the form of enzyme inhibitors (see
Chapter 8). The discovery of new drugs is greatly facilitated when a large number of potential pharmacophores can be simultaneously assayed in a
rapid, automated fashion—a process referred to as high­throughput screening (HTS). HTS employs robotics, optics, data processing, and
microfluidics to simultaneously conduct and monitor thousands of parallel enzyme assays. It thus serves as a perfect complement to combinatorial
chemistry, a method for generating large libraries of chemical compounds spanning all possible combinations of a given set of chemical precursors.
Enzyme­Linked Immunoassays
The sensitivity of enzyme assays can be exploited to detect proteins that lack catalytic activity. Enzyme­linked immunosorbent assays (ELISAs) use
antibodies covalently linked to a “reporter enzyme”, such as alkaline phosphatase or horseradish peroxidase, that can be used to produce easily­
detected chromogenic or fluorescent products in vitro. Serum or other biologic samples to be tested are first placed in multiwell microtiter plates.
Most proteins adhere to the plastic surface and thus are immobilized. Any exposed plastic that remains is subsequently “blocked” by adding a
nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added and the antibodies
allowed to adhere to any immobilized antigen molecules. Excess free antibody molecules are then removed by washing. The presence and quantity of
bound antibody is then determined by assaying the activity of the reporter enzyme, a quantity that should be proportional to the number of antibody
molecules present and, presumably, the antigen molecules to which they are bound.
NAD(P)+ ­Dependent Dehydrogenases Are Assayed Spectrophotometrically
The physicochemical properties of the reactants in an enzyme­catalyzed reaction dictate the options for the assay of enzyme activity.
Spectrophotometric assays exploit the ability of a substrate or product to absorb light. The reduced coenzymes NADH and NADPH, written as NAD(P)H,
absorb light at a wavelength of 340 nm, whereas their oxidized forms NAD(P)+ do not (Figure 7–10). When NAD(P)+ is reduced, the absorbance at 340
nm therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, when a dehydrogenase
catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 nm will be observed. In each case, the rate of change in absorbance at 340 nm will
be proportionate to the quantity of the enzyme present.
FIGURE 7–10
Absorption spectra of NAD+ and NADH. Densities are for a 44­mg/L solution in a cell with a 1­cm light path. NADP+ and NADPH have spectra
analogous to NAD+ and NADH, respectively.
The assay of enzymes whose reactions are not accompanied by a change in absorbance or fluorescence is generally more difficult. In some instances,
either the product or remaining substrate can be transformed into a more readily detected compound, although the reaction product may have to be
separated from unreacted substrate prior to measurement. An alternative strategy is to devise a synthetic substrate whose product absorbs light or
fluoresces. For example, hydrolysis of the phosphoester bond in p­nitrophenyl phosphate (pNPP), an artificial substrate molecule, is catalyzed at a
measurable rate
by numerous
phosphodiesterases, and serine proteases. While pNPP does not absorb visible light, the anionic form of
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Chapter
7: Enzymes:
of Action,
Peter J. Kennelly;
W.light
Rodwell
the
p­nitrophenol
(pKaMechanism
6.7) generated
on its hydrolysis
strongly Victor
absorbs
at 419 nm, and thus can be quantified.
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Many Enzymes May Be Assayed by Coupling to a Dehydrogenase
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School
Medicine
Public
Health
The assay of enzymes whose reactions are not accompanied by a change in absorbance or fluorescence is
generally
moreofdifficult.
In and
some
instances,
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either the product or remaining substrate can be transformed into a more readily detected compound, although
the reaction product may have to be
separated from unreacted substrate prior to measurement. An alternative strategy is to devise a synthetic substrate whose product absorbs light or
fluoresces. For example, hydrolysis of the phosphoester bond in p­nitrophenyl phosphate (pNPP), an artificial substrate molecule, is catalyzed at a
measurable rate by numerous phosphatases, phosphodiesterases, and serine proteases. While pNPP does not absorb visible light, the anionic form of
the p­nitrophenol (pKa 6.7) generated on its hydrolysis strongly absorbs light at 419 nm, and thus can be quantified.
Many Enzymes May Be Assayed by Coupling to a Dehydrogenase
Another quite general approach is to employ a “coupled” assay (Figure 7–11). Typically, a dehydrogenase whose substrate is the product of the
enzyme of interest is added in catalytic excess. The rate of appearance or disappearance of NAD(P)H then depends on the rate of the enzyme reaction
to which the dehydrogenase has been coupled.
FIGURE 7–11
Coupled enzyme assay for hexokinase activity. The production of glucose­6­phosphate by hexokinase is coupled to the oxidation of this product
by glucose­6­phosphate dehydrogenase in the presence of added enzyme and NADP+. When an excess of glucose­6­phosphate dehydrogenase is
present, the rate of formation of NADPH, which can be measured at 340 nm, is governed by the rate of formation of glucose­6­phosphate by
hexokinase.
THE ANALYSIS OF CERTAIN ENZYMES AIDS DIAGNOSIS
The analysis of enzymes in blood plasma plays a central role in the diagnosis of several disease processes. Many enzymes are functional constituents
of blood. Examples include pseudocholinesterase, lipoprotein lipase, and components of the cascades that trigger blood clotting, clot dissolution, and
opsonization of invading microbes. Several enzymes are released into plasma following cell death or injury. While these latter enzymes perform no
physiologic function in plasma, they can serve as biomarkers, molecules whose appearance or levels can assist in the diagnosis and prognosis of
diseases and injuries affecting specific tissues. The plasma concentration of an enzyme or other protein released consequent to injury may rise early
or late, and may decline rapidly or slowly. Cytoplasmic proteins tend to appear more rapidly than those from subcellular organelles.
Quantitative analysis of the activity of released enzymes or other proteins, typically in plasma or serum but also in urine or various cells, provides
information concerning diagnosis, prognosis, and response to treatment. Assays of enzyme activity typically employ standard kinetic assays of initial
reaction rates. Table 7–1 lists several enzymes of value in clinical diagnosis. Note that these enzymes are not absolutely specific for the indicated
disease. For example, elevated blood levels of prostatic acid phosphatase are associated typically with prostate cancer, but also may occur with certain
other cancers and noncancerous conditions. Interpretation of enzyme assay data must make due allowance for the sensitivity and the diagnostic
specificity of the enzyme test, together with other factors elicited through a comprehensive clinical examination that includes patient’s age, sex, prior
history, and possible drug use.
TABLE 7–1
Principal Serum Enzymes Used in Clinical Diagnosis
Serum Enzyme
Major Diagnostic Use
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Alanine aminotransferase
Viral hepatitis
Chapter 7: Enzymes: Mechanism of Action, Peter J. Kennelly; Victor W. Rodwell
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Amylase
Acute pancreatitis
Page 13 / 19
disease. For example, elevated blood levels of prostatic acid phosphatase are associated typically with prostate cancer, but also may occur with certain
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other cancers and noncancerous conditions. Interpretation of enzyme assay data must make due allowance for the sensitivity and the diagnostic
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specificity of the enzyme test, together with other factors elicited through a comprehensive clinical examination that includes patient’s age, sex, prior
history, and possible drug use.
TABLE 7–1
Principal Serum Enzymes Used in Clinical Diagnosis
Serum Enzyme
Major Diagnostic Use
Alanine aminotransferase (ALT)
Viral hepatitis
Amylase
Acute pancreatitis
Ceruloplasmin
Hepatolenticular degeneration (Wilson disease)
Creatine kinase
Muscle disorders
γ­Glutamyl transferase
Various liver diseases
Lactate dehydrogenase isozyme 5
Liver diseases
Lipase
Acute pancreatitis
β­Glucocerebrosidase
Gaucher disease
Phosphatase, acid
Prostate disease, including cancer
Phosphatase, alkaline (isozymes)
Various bone disorders, obstructive liver diseases
ALT, alanine aminotransferase.
Note: Many of the above enzymes are not specific to the disease listed.
Analysis of Serum Enzymes Following Tissue Injury
An enzyme useful for diagnostic enzymology should be relatively specific for the tissue or organ under study, and should appear in the plasma or other
fluid at a time useful for diagnosis (the “diagnostic window”). In the case of a myocardial infarction (MI), detection must be possible within a few hours
or less of a preliminary diagnosis to permit initiation of appropriate therapy. The first enzymes used to diagnose MI were aspartate aminotransferase
(AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH). Diagnosis using LDH exploits the tissue­specific variations in its quaternary
structure (Figure 7–12). However, it is released relatively slowly following injury. Creatine kinase (CK) has three tissue­specific isozymes: CK­MM
(skeletal muscle), CK­BB (brain), and CK­MB (heart and skeletal muscle), along with a more optimal diagnostic window. As with LDH, individual CK
isozymes are separable by electrophoresis. Today, assays of plasma CK levels are primarily used to assess skeletal muscle disorders such as Duchene
muscular dystrophy.
FIGURE 7–12
Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in human serum. Samples of serum were separated by
electrophoresis. LDH isozymes were then visualized using a dye­coupled reaction­specific for LDH. Pattern A is serum from a patient with a myocardial
infarct; B is normal serum; and C is serum from a patient with liver disease. Arabic numerals identify LDH isozymes 1 through 5. Electrophoresis and a
specific detection technique thus can be used to visualize isozymes of enzymes other than LDH.
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Chapter 7: Enzymes: Mechanism of Action, Peter J. Kennelly; Victor W. Rodwell
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Page 14 / 19
Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in human serum. Samples of serum were separated by
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electrophoresis. LDH isozymes were then visualized using a dye­coupled reaction­specific for LDH. Pattern A is serum from a patient with a myocardial
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infarct; B is normal serum; and C is serum from a patient with liver disease. Arabic numerals identify LDH isozymes 1 through 5. Electrophoresis and a
specific detection technique thus can be used to visualize isozymes of enzymes other than LDH.
Plasma Troponin Constitutes the Currently Preferred Diagnostic Marker for an MI
Troponin is a complex of three proteins present in the contractile apparatus of skeletal and cardiac muscle but not in smooth muscle (see Chapter
51). Troponin levels in plasma typically rise for 2 to 6 hours after an MI, and remain elevated for 4 to 10 days. Immunological measurements of plasma
levels of cardiac troponins I and T thus provide sensitive and specific indicators of damage to heart muscle. Since other sources of heart muscle
damage also elevate serum troponin levels, cardiac troponins provide a general marker of cardiac injury.
Additional Clinical Uses of Enzymes
Enzymes are employed in the clinical laboratory to determine the presence and the concentration of critical metabolites. For example, glucose oxidase
frequently is utilized to measure plasma glucose concentration. Enzymes also are employed with increasing frequency for the treatment of injury and
disease. Examples include tissue plasminogen activator (tPA) or streptokinase for treatment of acute MI, and trypsin for treatment of cystic fibrosis.
Intravenous infusion of recombinantly produced glycosylases can be used to treat lysosomal storage syndromes such as Gaucher disease (β­
glucosidase), Pompe disease (α­glucosidase), Fabry disease (α­galactosidase A), Sly disease (β­glucuronidase), and mucopolysaccharidosis types I, II,
and VI—also known as Hurler syndrome (α­L­iduronidase), Hunter syndrome (iduronate 2­sulfatase), and Maroteaux­Lamy syndrome (arylsulfatase B),
respectively.
ENZYMES FACILITATE DIAGNOSIS OF GENETIC & INFECTIOUS DISEASES
Many diagnostic techniques take advantage of the specificity and efficiency of the enzymes that act on oligonucleotides such as DNA. Early on
restriction endonucleases emerged as an important tool for both clinical and forensic analyses. Restriction endonucleases, often referred to as
restriction enzymes, cleave double­stranded DNA at sites specified by a sequence of four, six, or more base pairs called restriction sites (see Chapter
39). If an individual bears a mutation or polymorphism that either eliminates or generates the restriction site for one of the endonucleases being used,
the number and size of the DNA fragments produced on cleavage will differ from that of another individual. Such restriction fragment length
polymorphisms (RFLPs) can be used for prenatal detection of deleterious mutations characteristic of hereditary disorders such as sickle cell trait, β­
thalassemia, infant phenylketonuria, and Huntington disease. They also can be used as molecular biological “fingerprints” to help trace the source of a
sample of DNA to a specific individual. RFLP suffers from several important limitations, the most critical of which was the requirement for relatively
high quantities of DNA to employ as substrates for the restriction enzymes. Today, a dramatically more sensitive set of molecular diagnostic tools
based on the polymerase chain reaction (PCR) have largely supplanted RFLP analysis.
Medical Applications of the Polymerase Chain Reaction
As described in Chapter 39, the polymerase chain reaction (PCR) employs a thermostable DNA polymerase and appropriate oligonucleotide primers to
produce thousands of copies of a defined segment of DNA from a minute quantity of starting material. PCR enables medical, biological, and forensic
scientists to detect and characterize DNA present initially at levels too low for direct detection. In addition to screening for genetic mutations, PCR can
be used to detect pathogens and parasites such as Trypanosoma cruzi, the causative agent of Chagas disease, and Neisseria meningitides, the
causative agent of bacterial meningitis, through the selective amplification of their DNA.
RECOMBINANT DNA PROVIDES AN IMPORTANT TOOL FOR STUDYING ENZYMES
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7: Enzymes:
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Action,
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Kennelly;
W. Rodwell
Highly purified
samples
of enzymesofare
essential
forJ.the
study ofVictor
their structure
and function. The isolation of an individual enzyme, particularly
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present in low concentration, from among the thousands of proteins present in a cell can be extremely difficult. By cloning the gene for the enzyme of
interest, it generally is possible to produce large quantities of its encoded protein in Escherichia coli or yeast. However, not all animal proteins can be
produce thousands of copies of a defined segment of DNA from a minute quantity of starting material. PCR enables medical, biological, and forensic
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scientists to detect and characterize DNA present initially at levels too low for direct detection. In addition to screening for genetic mutations, PCR can
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be used to detect pathogens and parasites such as Trypanosoma cruzi, the causative agent of Chagas disease, and Neisseria meningitides, the
causative agent of bacterial meningitis, through the selective amplification of their DNA.
RECOMBINANT DNA PROVIDES AN IMPORTANT TOOL FOR STUDYING ENZYMES
Highly purified samples of enzymes are essential for the study of their structure and function. The isolation of an individual enzyme, particularly one
present in low concentration, from among the thousands of proteins present in a cell can be extremely difficult. By cloning the gene for the enzyme of
interest, it generally is possible to produce large quantities of its encoded protein in Escherichia coli or yeast. However, not all animal proteins can be
expressed in their appropriately folded, functionally competent form in microbial cells as these organisms cannot perform certain posttranslational
processing tasks specific to higher organisms. In these instances, options include expression of recombinant genes in cultured animal cell systems or
by employing the baculovirus expression vector of cultured insect cells. For more details concerning recombinant DNA techniques, see Chapter 39.
Recombinant Fusion Proteins Are Purified by Affinity Chromatography
Recombinant DNA technology can also be used to generate proteins specifically modified to render them readily purified by affinity chromatography.
The gene of interest is linked to an additional oligonucleotide sequence that encodes a carboxyl or amino terminal extension to the protein of interest.
The resulting fusion protein contains a new domain tailored to interact with an appropriately modified affinity support. One popular approach is to
attach an oligonucleotide that encodes six consecutive histidine residues. The expressed “His tag” protein binds to chromatographic supports that
contain an immobilized divalent metal ion such as Ni2+ or Cd2+. This approach exploits the ability of these divalent cations to bind His residues. Once
bound, contaminating proteins are washed off and the His­tagged enzyme is eluted with buffers containing high concentrations of free histidine or
imidazole, which compete with the polyhistidine tails for binding to the immobilized metal ions. Alternatively, the substrate­binding domain of
glutathione S­transferase (GST) can serve as a “GST tag.” Figure 7–13 illustrates the purification of a GST­fusion protein using an affinity support
containing bound glutathione.
FIGURE 7–13
Use of glutathione S­transferase (GST) fusion proteins to purify recombinant proteins. (GSH, glutathione.)
The addition of an N­terminal fusion domain may also help induce proper folding of the remainder of the recombinant polypeptide. Most fusion
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domains also possess a cleavage site for a highly specific protease such as thrombin in the region that links the two portions of the proteinPage
to permit
its
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Chapter 7: Enzymes: Mechanism of Action, Peter J. Kennelly; Victor W. Rodwell
eventual
removal.
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Site­Directed Mutagenesis Provides Mechanistic Insights
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The addition of an N­terminal fusion domain may also help induce proper folding of the remainder of the recombinant polypeptide. Most fusion
domains also possess a cleavage site for a highly specific protease such as thrombin in the region that links the two portions of the protein to permit its
eventual removal.
Site­Directed Mutagenesis Provides Mechanistic Insights
Once the ability to express a protein from its cloned gene has been established, it is possible to employ site­directed mutagenesis to change
specific aminoacyl residues by altering their codons. Used in combination with kinetic analyses and x­ray crystallography, this approach facilitates
identification of the specific roles of given aminoacyl residues in substrate binding and catalysis. For example, the inference that a particular aminoacyl
residue functions as a general acid can be tested by replacing it with an aminoacyl residue incapable of donating a proton.
RIBOZYMES: ARTIFACTS FROM THE RNA WORLD
Cech Discovered the First Catalytic RNA Molecule
For many years after their discovery it was assumed that all enzymes were proteins. However, while examining the processing of ribosomal RNA (rRNA)
molecules in the ciliated protozoan Tetrahymena in the early 1980s, Thomas Cech and his coworkers observed that processing of the 26S rRNA
proceeded smoothly in vitro even in the total absence of protein. They subsequently traced the source of this splicing activity to a 413 bp catalytic
segment of the RNA, which they termed a ribozyme (see Chapter 36)—a discovery that earned Cech a Nobel Prize.
Several other ribozymes have since been discovered. The vast majority catalyze nucleophilic displacement reactions that target the phosphodiester
bonds of the RNA backbone. In small self­cleaving RNAs, such as hammerhead or hepatitis delta virus RNA, the attacking nucleophile is water and the
result is hydrolysis. For the large group I intron ribozymes, the attacking nucleophile is the 3′­hydroxyl of the terminal ribose of another segment of
RNA and the result is a splicing reaction.
The Ribosome—The Ultimate Ribozyme
The ribosome was the first example of a “molecular machine” to be recognized. A massive complex comprised of scores of protein subunits and
several large ribosomal RNA molecules, the ribosome performs the vitally important and highly complex process of synthesizing long polypeptide
chains following the instructions encoded in messenger RNA (mRNA) molecules (see Chapter 37). For many years, it was assumed that rRNAs played a
passive, structural role, or perhaps assisted in the recognition of cognate mRNAs through a base pairing mechanism. It was thus somewhat surprising
when it was discovered that rRNAs were both necessary and sufficient for catalyzing peptide synthesis.
The RNA World Hypothesis
The discovery of ribozymes has had a profound influence on evolutionary theory. For many years, scientists had hypothesized that the first biologic
catalysts were formed when amino acids contained in the primordial soup coalesced to form simple proteins. With the realization that RNA could both
carry information and catalyze chemical reactions, a new “RNA World” hypothesis emerged in which RNA constituted the first biologic macromolecule.
Eventually, a more chemically stable oligonucleotide, DNA, superseded RNA for long­term information storage, while proteins, by virtue of their greater
chemical functional group and conformational diversity dominated catalysis. If one assumes that some sort of RNA­protein hybrid was formed as an
intermediate in the transition from ribonucleotide to polypeptide catalysts, one needs to look no further than the ribosome to find the presumed
missing link.
Why did not proteins take over all catalytic functions? Presumably, in the case of the ribosome the process was both too complex and too essential to
permit much opportunity for possible competitors to gain a foothold. In the case of the small self­cleaving RNAs and self­splicing introns, they may
represent one of the few cases in which RNA autocatalysis is more efficient than development of a new protein catalyst.
SUMMARY
Enzymes are efficient catalysts whose stringent specificity extends to the kind of reaction catalyzed, and typically, to the stereochemistry of their
substrates.
In many enzymes,
suite
chemical
tools available to facilitate catalysis has been expanded using non­amino acid molecules. When tightly
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bound,
these inorganic
and organic
molecules
referredVictor
to as prosthetic
groups. If loosely bound (dissociable), they are referred to
as 17 / 19
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7: Enzymes:
Mechanism
of Action,
Peter J.are
Kennelly;
W. Rodwell
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cofactors.
Coenzymes, many of which are derivatives of B vitamins, serve as “shuttles” for commonly used groups such as amines, electrons, and acetyl
SUMMARY
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Enzymes are efficient catalysts whose stringent specificity extends to the kind of reaction catalyzed, and typically, to the stereochemistry of their
substrates.
In many enzymes, the suite of chemical tools available to facilitate catalysis has been expanded using non­amino acid molecules. When tightly
bound, these inorganic and organic molecules are referred to as prosthetic groups. If loosely bound (dissociable), they are referred to as
cofactors.
Coenzymes, many of which are derivatives of B vitamins, serve as “shuttles” for commonly used groups such as amines, electrons, and acetyl
groups.
During catalysis, enzymes redirect the conformational changes induced by substrate binding to effect complementary changes that facilitate
transformation into product.
Mechanistic strategies employed by enzymes to facilitate catalysis include the introduction of strain, approximation of reactants, acid–base
catalysis, and covalent catalysis.
Aminoacyl residues that participate in catalysis are highly conserved through the evolution of enzymes.
The ability to change residues suspected of being important in catalysis or substrate binding by site­directed mutagenesis provides important
insights into mechanisms of enzyme action.
The catalytic activity of enzymes reveals their presence, facilitates their detection, and provides the basis for enzyme­linked immunoassays.
Many enzymes can be assayed spectrophotometrically by coupling them to an NAD(P)H­dependent dehydrogenase.
Assay of plasma proteins, including several enzymes, aids clinical diagnosis and prognosis.
Restriction endonucleases facilitate diagnosis of genetic diseases by revealing restriction fragment length polymorphisms.
The PCR amplifies DNA initially present in quantities too small for analysis.
Attachment of a polyhistidyl, GST, or other “tag” to the N­ or C­terminus of a recombinant protein facilitates its purification by affinity
chromatography.
Not all enzymes are proteins. Several ribozymes are known that can cut and resplice the phosphodiester bonds of RNA while it is the RNA
components of the ribosome that are primarily responsible for catalyzing polypeptide synthesis.
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Sanabria H, Rodnin D, Hemmen K, et al: Resolving dynamics and function of transient states in single enzyme molecules. Nature Commun
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Chapter 7: Enzymes: Mechanism of Action, Peter J. Kennelly; Victor W. Rodwell
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