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Chapters derived from:

The Basics of General, Organic, and Biological Chemistry

By David W. Ball, John W. Hill, and Rhonda J. Scott


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Attribution-NonCommercial-ShareAlikeCC BY-NC-SA


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Learning Objectives

Describe the four levels of protein structure.Identify the types of attractive interactions that hold proteins in their most stable three-dimensional structure.Explain what happens when proteins are denatured.Identify how a protein can be denatured.

Each of the thousands of naturally occurring proteins has its own characteristic amino acid composition and sequence that result in a unique three-dimensional shape. Since the 1950s, scientists have determined the amino acid sequences and three-dimensional conformation of numerous proteins and thus obtained important clues on how each protein performs its specific function in the body.

Proteins are compounds of high molar mass consisting largely or entirely of chains of amino acids. Because of their great complexity, protein molecules cannot be classified on the basis of specific structural similarities, as carbohydrates and lipids are categorized. The two major structural classifications of proteins are based on far more general qualities: whether the protein is (1) fiberlike and insoluble or (2) globular and soluble. Some proteins, such as those that compose hair, skin, muscles, and connective tissue, are fiberlike. These fibrous proteins are insoluble in water and usually serve structural, connective, and protective functions. Examples of fibrous proteins are keratins, collagens, myosins, and elastins. Hair and the outer layer of skin are composed of keratin. Connective tissues contain collagen. Myosins are muscle proteins and are capable of contraction and extension. Elastins are found in ligaments and the elastic tissue of artery walls.

Globular proteins, the other major class, are soluble in aqueous media. In these proteins, the chains are folded so that the molecule as a whole is roughly spherical. Familiar examples include egg albumin from egg whites and serum albumin in blood. Serum albumin plays a major role in transporting fatty acids and maintaining a proper balance of osmotic pressures in the body. Hemoglobin and myoglobin, which are important for binding oxygen, are also globular proteins.


Levels of Protein Structure

The structure of proteins is generally described as having four organizational levels. The first of these is the primary structure, which is the number and sequence of amino acids in a protein’s polypeptide chain or chains, beginning with the free amino group and maintained by the peptide bonds connecting each amino acid to the next. The primary structure of insulin, composed of 51 amino acids, is shown in Figure 9.1 "Primary Structure of Human Insulin".


Figure 9.1 Primary Structure of Human Insulin

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Human insulin, whose amino acid sequence is shown here, is a hormone that is required for the proper metabolism of glucose.


A protein molecule is not a random tangle of polypeptide chains. Instead, the chains are arranged in unique but specific conformations. The term secondary structure refers to the fixed arrangement of the polypeptide backbone. On the basis of X ray studies, Linus Pauling and Robert Corey postulated that certain proteins or portions of proteins twist into a spiral or a helix. This helix is stabilized by intrachain hydrogen bonding between the carbonyl oxygen atom of one amino acid and the amide hydrogen atom four amino acids up the chain (located on the next turn of the helix) and is known as a right-handed α-helix. X ray data indicate that this helix makes one turn for every 3.6 amino acids, and the side chains of these amino acids project outward from the coiled backbone (Figure 9.2 "A Ball-and-Stick Model of an α-Helix"). The α-keratins, found in hair and wool, are exclusively α-helical in conformation. Some proteins, such as gamma globulin, chymotrypsin, and cytochrome c, have little or no helical structure. Others, such as hemoglobin and myoglobin, are helical in certain regions but not in others.


Figure 9.2 A Ball-and-Stick Model of an α-Helix

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This ball-and-stick model shows the intrachain hydrogen bonding between carbonyl oxygen atoms and amide hydrogen atoms. Each turn of the helix spans 3.6 amino acids. Note that the side chains (represented as green spheres) point out from the helix.


Another common type of secondary structure, called the β-pleated sheet conformation, is a sheetlike arrangement in which two or more extended polypeptide chains (or separate regions on the same chain) are aligned side by side. The aligned segments can run either parallel or antiparallel—that is, the N-terminals can face in the same direction on adjacent chains or in different directions—and are connected by interchain hydrogen bonding (Figure 9.3 "A Ball-and-Stick Model of the β-Pleated Sheet Structure in Proteins"). The β-pleated sheet is particularly important in structural proteins, such as silk fibroin. It is also seen in portions of many enzymes, such as carboxypeptidase A and lysozyme.


Figure 9.3 A Ball-and-Stick Model of the β-Pleated Sheet Structure in Proteins

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The side chains extend above or below the sheet and alternate along the chain. The protein chains are held together by interchain hydrogen bonding.


Tertiary structure refers to the unique three-dimensional shape of the protein as a whole, which results from the folding and bending of the protein backbone. The tertiary structure is intimately tied to the proper biochemical functioning of the protein. Figure 9.4 "A Ribbon Model of the Three-Dimensional Structure of Insulin" shows a depiction of the three-dimensional structure of insulin.


Figure 9.4 A Ribbon Model of the Three-Dimensional Structure of Insulin

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The spiral regions represent sections of the polypeptide chain that have an α-helical structure, while the broad arrows represent β-pleated sheet structures.


Four major types of attractive interactions determine the shape and stability of the tertiary structure of proteins.

1. Ionic bonding. Ionic bonds result from electrostatic attractions between positively and negatively charged side chains of amino acids. For example, the mutual attraction between an aspartic acid carboxylate ion and a lysine ammonium ion helps to maintain a particular folded area of a protein (part (a) of Figure 9.5 "Tertiary Protein Structure Interactions").

2. Hydrogen bonding. Hydrogen bonding forms between a highly electronegative oxygen atom or a nitrogen atom and a hydrogen atom attached to another oxygen atom or a nitrogen atom, such as those found in polar amino acid side chains. Hydrogen bonding (as well as ionic attractions) is extremely important in both the intra- and intermolecular interactions of proteins (part (b) of Figure 9.5 "Tertiary Protein Structure Interactions").

3. Disulfide linkages. Two cysteine amino acid units may be brought close together as the protein molecule folds. Subsequent oxidation and linkage of the sulfur atoms in the highly reactive sulfhydryl (SH) groups leads to the formation of cystine (part (c) of Figure 9.5 "Tertiary Protein Structure Interactions"). Intrachain disulfide linkages are found in many proteins, including insulin (yellow bars in Figure 9.1 "Primary Structure of Human Insulin") and have a strong stabilizing effect on the tertiary structure.


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4. Dispersion forces. Dispersion forces arise when a normally nonpolar atom becomes momentarily polar due to an uneven distribution of electrons, leading to an instantaneous dipole that induces a shift of electrons in a neighboring nonpolar atom. Dispersion forces are weak but can be important when other types of interactions are either missing or minimal (part (d) of Figure 9.5 "Tertiary Protein Structure Interactions"). This is the case with fibroin, the major protein in silk, in which a high proportion of amino acids in the protein have nonpolar side chains. The term hydrophobic interaction is often misused as a synonym for dispersion forces. Hydrophobic interactions arise because water molecules engage in hydrogen bonding with other water molecules (or groups in proteins capable of hydrogen bonding). Because nonpolar groups cannot engage in hydrogen bonding, the protein folds in such a way that these groups are buried in the interior part of the protein structure, minimizing their contact with water.


Figure 9.5 Tertiary Protein Structure Interactions

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Four interactions stabilize the tertiary structure of a protein: (a) ionic bonding, (b) hydrogen bonding, (c) disulfide linkages, and (d) dispersion forces.


When a protein contains more than one polypeptide chain, each chain is called a subunit. The arrangement of multiple subunits represents a fourth level of structure, the quaternary structure of a protein. Hemoglobin, with four polypeptide chains or subunits, is the most frequently cited example of a protein having quaternary structure (Figure 9.6 "The Quaternary Structure of Hemoglobin"). The quaternary structure of a protein is produced and stabilized by the same kinds of interactions that produce and maintain the tertiary structure. A schematic representation of the four levels of protein structure is in Figure 9.7 "Levels of Structure in Proteins".


Figure 9.6 The Quaternary Structure of Hemoglobin

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Hemoglobin is a protein that transports oxygen throughout the body.


Source: Image from the RCSB PDB (www.pdb.org) of PDB ID 1I3D (R.D. Kidd, H.M. Baker, A.J. Mathews, T. Brittain, E.N. Baker (2001) Oligomerization and ligand binding in a homotetrameric hemoglobin: two high-resolution crystal structures of hemoglobin Bart's (gamma(4)), a marker for alpha-thalassemia. Protein Sci. 1739–1749).


Figure 9.7 Levels of Structure in Proteins

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The primary structure consists of the specific amino acid sequence. The resulting peptide chain can twist into an α-helix, which is one type of secondary structure. This helical segment is incorporated into the tertiary structure of the folded polypeptide chain. The single polypeptide chain is a subunit that constitutes the quaternary structure of a protein, such as hemoglobin that has four polypeptide chains.


Denaturation of Proteins

The highly organized structures of proteins are truly masterworks of chemical architecture. But highly organized structures tend to have a certain delicacy, and this is true of proteins. Denaturation is the term used for any change in the three-dimensional structure of a protein that renders it incapable of performing its assigned function. A denatured protein cannot do its job. (Sometimes denaturation is equated with the precipitation or coagulation of a protein; our definition is a bit broader.) A wide variety of reagents and conditions, such as heat, organic compounds, pH changes, and heavy metal ions can cause protein denaturation (Table 9.1 "Protein Denaturation Methods").


Table 9.1 Protein Denaturation Methods

MethodEffect on Protein Structure
Heat above 50°C or ultraviolet (UV) radiationHeat or UV radiation supplies kinetic energy to protein molecules, causing their atoms to vibrate more rapidly and disrupting relatively weak hydrogen bonding and dispersion forces.
Use of organic compounds, such as ethyl alcoholThese compounds are capable of engaging in intermolecular hydrogen bonding with protein molecules, disrupting intramolecular hydrogen bonding within the protein.
Salts of heavy metal ions, such as mercury, silver, and leadThese ions form strong bonds with the carboxylate anions of the acidic amino acids or SH groups of cysteine, disrupting ionic bonds and disulfide linkages.
Alkaloid reagents, such as tannic acid (used in tanning leather)These reagents combine with positively charged amino groups in proteins to disrupt ionic bonds.

Anyone who has fried an egg has observed denaturation. The clear egg white turns opaque as the albumin denatures and coagulates. No one has yet reversed that process. However, given the proper circumstances and enough time, a protein that has unfolded under sufficiently gentle conditions can refold and may again exhibit biological activity (Figure 9.8 "Denaturation and Renaturation of a Protein"). Such evidence suggests that, at least for these proteins, the primary structure determines the secondary and tertiary structure. A given sequence of amino acids seems to adopt its particular three-dimensional arrangement naturally if conditions are right.


Figure 9.8 Denaturation and Renaturation of a Protein

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The denaturation (unfolding) and renaturation (refolding) of a protein is depicted. The red boxes represent stabilizing interactions, such as disulfide linkages, hydrogen bonding, and/or ionic bonds.


The primary structures of proteins are quite sturdy. In general, fairly vigorous conditions are needed to hydrolyze peptide bonds. At the secondary through quaternary levels, however, proteins are quite vulnerable to attack, though they vary in their vulnerability to denaturation. The delicately folded globular proteins are much easier to denature than are the tough, fibrous proteins of hair and skin.


What is the predominant attractive force that stabilizes the formation of secondary structure in proteins?


Tertiary structure refers to the unique three-dimensional shape of a single polypeptide chain, while quaternary structure describes the interaction between multiple polypeptide chains for proteins that have more than one polypeptide chain.


(1) heat a protein above 50°C or expose it to UV radiation; (2) add organic solvents, such as ethyl alcohol, to a protein solution; (3) add salts of heavy metal ions, such as mercury, silver, or lead; and (4) add alkaloid reagents such as tannic acid


Key Takeaways

Proteins can be divided into two categories: fibrous, which tend to be insoluble in water, and globular, which are more soluble in water.A protein may have up to four levels of structure. The primary structure consists of the specific amino acid sequence. The resulting peptide chain can form an α-helix or β-pleated sheet (or local structures not as easily categorized), which is known as secondary structure. These segments of secondary structure are incorporated into the tertiary structure of the folded polypeptide chain. The quaternary structure describes the arrangements of subunits in a protein that contains more than one subunit.Four major types of attractive interactions determine the shape and stability of the folded protein: ionic bonding, hydrogen bonding, disulfide linkages, and dispersion forces.A wide variety of reagents and conditions can cause a protein to unfold or denature.

Exercises

1. Classify each protein as fibrous or globular.

a. albumin

b. myosin

c. fibroin

2. Classify each protein as fibrous or globular.

a. hemoglobin

b. keratin

c. myoglobin

3. What name is given to the predominant secondary structure found in silk?

4. What name is given to the predominant secondary structure found in wool protein?

5. A protein has a tertiary structure formed by interactions between the side chains of the following pairs of amino acids. For each pair, identify the strongest type of interaction between these amino acids.

a. aspartic acid and lysine

b. phenylalanine and alanine

c. serine and lysine

d. two cysteines

6. A protein has a tertiary structure formed by interactions between the side chains of the following pairs of amino acids. For each pair, identify the strongest type of interaction between these amino acids.

a. valine and isoleucine

b. asparagine and serine

c. glutamic acid and arginine

d. tryptophan and methionine

7. What level(s) of protein structure is(are) ordinarily disrupted in denaturation? What level(s) is(are) not?

8. Which class of proteins is more easily denatured—fibrous or globular?


Answers

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a. globular

b. fibrous

c. fibrous

3. β-pleated sheet

5.

a. ionic bonding

b. dispersion forces

c. dispersion forces

d. disulfide linkage

7. Protein denaturation disrupts the secondary, tertiary, and quaternary levels of structure. Only primary structure is unaffected by denaturation.


A catalyst is any substance that increases the rate or speed of a chemical reaction without being changed or consumed in the reaction. Enzymes are biological catalysts, and nearly all of them are proteins. The reaction rates attained by enzymes are truly amazing. In their presence, reactions occur at rates that are a million (106) or more times faster than would be attainable in their absence. What is even more amazing is that enzymes perform this function at body temperature (~37°C) and physiological pH (pH ~7), rather than at the conditions that are typically necessary to increase reaction rates (high temperature or pressure, the use of strong oxidizing or reducing agents or strong acids or bases, or a combination of any of these). In addition, enzymes are highly specific in their action; that is, each enzyme catalyzes only one type of reaction in only one compound or a group of structurally related compounds. The compound or compounds on which an enzyme acts are known as its substrates.

Hundreds of enzymes have been purified and studied in an effort to understand how they work so effectively and with such specificity. The resulting knowledge has been used to design drugs that inhibit or activate particular enzymes. An example is the intensive research to improve the treatment of or find a cure for acquired immunodeficiency syndrome (AIDS). AIDS is caused by the human immunodeficiency virus (HIV). Researchers are studying the enzymes produced by this virus and are developing drugs intended to block the action of those enzymes without interfering with enzymes produced by the human body. Several of these drugs have now been approved for use by AIDS patients.

The first enzymes to be discovered were named according to their source or method of discovery. The enzyme pepsin, which aids in the hydrolysis of proteins, is found in the digestive juices of the stomach (Greek pepsis, meaning “digestion”). Papain, another enzyme that hydrolyzes protein (in fact, it is used in meat tenderizers), is isolated from papayas. As more enzymes were discovered, chemists recognized the need for a more systematic and chemically informative identification scheme.

In the current numbering and naming scheme, under the oversight of the Nomenclature Commission of the International Union of Biochemistry, enzymes are arranged into six groups according to the general type of reaction they catalyze (Table 9.2 "Classes of Enzymes"), with subgroups and secondary subgroups that specify the reaction more precisely. Each enzyme is assigned a four-digit number, preceded by the prefix EC—for enzyme classification—that indicates its group, subgroup, and so forth. This is demonstrated in Table 9.3 "Assignment of an Enzyme Classification Number" for alcohol dehydrogenase. Each enzyme is also given a name consisting of the root of the name of its substrate or substrates and the -ase suffix. Thus urease is the enzyme that catalyzes the hydrolysis of urea.


Table 9.2 Classes of Enzymes

ClassType of Reaction CatalyzedExamples
oxidoreductasesoxidation-reduction reactionsDehydrogenases catalyze oxidation-reduction reactions involving hydrogen and reductases catalyze reactions in which a substrate is reduced.
transferasestransfer reactions of groups, such as methyl, amino, and acetylTransaminases catalyze the transfer of amino group, and kinases catalyze the transfer of a phosphate group.
hydrolaseshydrolysis reactionsLipases catalyze the hydrolysis of lipids, and proteases catalyze the hydrolysis of proteins
lyasesreactions in which groups are removed without hydrolysis or addition of groups to a double bondDecarboxylases catalyze the removal of carboxyl groups.
isomerasesreactions in which a compound is converted to its isomerIsomerases may catalyze the conversion of an aldose to a ketose, and mutases catalyze reactions in which a functional group is transferred from one atom in a substrate to another.
ligasesreactions in which new bonds are formed between carbon and another atom; energy is requiredSynthetases catalyze reactions in which two smaller molecules are linked to form a larger one.

Table 9.3 Assignment of an Enzyme Classification Number

Alcohol Dehydrogenase: EC 1.1.1.1
The first digit indicates that this enzyme is an oxidoreductase; that is, an enzyme that catalyzes an oxidation-reduction reaction.
The second digit indicates that this oxidoreductase catalyzes a reaction involving a primary or secondary alcohol.
The third digit indicates that either the coenzyme NAD+ or NADP+ is required for this reaction.
The fourth digit indicates that this was the first enzyme isolated, characterized, and named using this system of nomenclature.
The systematic name for this enzyme is alcohol:NAD+ oxidoreductase, while the recommended or common name is alcohol dehydrogenase.
Reaction catalyzed:
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Concept Review Exercise

In the small intestine, sucrose is hydrolyzed to form glucose and fructose in a reaction catalyzed by sucrase.


Key Takeaways

An enzyme is a biological catalyst, a substance that increases the rate of a chemical reaction without being changed or consumed in the reaction.A systematic process is used to name and classify enzymes.

Exercises

1. Identify the substrate catalyzed by each enzyme.

a. lactase

b. cellulase

c. peptidase

2. Identify the substrate catalyzed by each enzyme.

a. lipase

b. amylase

c. maltase

3. Identify each type of enzyme.

a. decarboxylase

b. protease

c. transaminase

4. Identify each type of enzyme.

a. dehydrogenase

b. isomerase

c. lipase


Answers

1.

a. lactose

b. cellulose

c. peptides

3.

a. lyase

b. hydrolase

c. transferase


9.3 Enzyme Action


Learning Objective

Describe the interaction between an enzyme and its substrate.

Enzyme-catalyzed reactions occur in at least two steps. In the first step, an enzyme molecule (E) and the substrate molecule or molecules (S) collide and react to form an intermediate compound called the enzyme-substrate (E–S) complex. (This step is reversible because the complex can break apart into the original substrate or substrates and the free enzyme.) Once the E–S complex forms, the enzyme is able to catalyze the formation of product (P), which is then released from the enzyme surface:

S + E → E–S E–S → P + E

Hydrogen bonding and other electrostatic interactions hold the enzyme and substrate together in the complex. The structural features or functional groups on the enzyme that participate in these interactions are located in a cleft or pocket on the enzyme surface. This pocket, where the enzyme combines with the substrate and transforms the substrate to product is called the active site of the enzyme (Figure 9.9 "Substrate Binding to the Active Site of an Enzyme"). It possesses a unique conformation (including correctly positioned bonding groups) that is complementary to the structure of the substrate, so that the enzyme and substrate molecules fit together in much the same manner as a key fits into a tumbler lock. In fact, an early model describing the formation of the enzyme-substrate complex was called the lock-and-key model (Figure 9.10 "The Lock-and-Key Model of Enzyme Action"). This model portrayed the enzyme as conformationally rigid and able to bond only to substrates that exactly fit the active site.


Figure 9.9 Substrate Binding to the Active Site of an Enzyme

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The enzyme dihydrofolate reductase is shown with one of its substrates: NADP+ (a) unbound and (b) bound. The NADP+ (shown in red) binds to a pocket that is complementary to it in shape and ionic properties.


Figure 9.10 The Lock-and-Key Model of Enzyme Action

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(a) Because the substrate and the active site of the enzyme have complementary structures and bonding groups, they fit together as a key fits a lock. (b) The catalytic reaction occurs while the two are bonded together in the enzyme-substrate complex.


Working out the precise three-dimensional structures of numerous enzymes has enabled chemists to refine the original lock-and-key model of enzyme actions. They discovered that the binding of a substrate often leads to a large conformational change in the enzyme, as well as to changes in the structure of the substrate or substrates. The current theory, known as the induced-fit model, says that enzymes can undergo a change in conformation when they bind substrate molecules, and the active site has a shape complementary to that of the substrate only after the substrate is bound, as shown for hexokinase in Figure 9.11 "The Induced-Fit Model of Enzyme Action". After catalysis, the enzyme resumes its original structure.


Figure 9.11 The Induced-Fit Model of Enzyme Action

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(a) The enzyme hexokinase without its substrate (glucose, shown in red) is bound to the active site. (b) The enzyme conformation changes dramatically when the substrate binds to it, resulting in additional interactions between hexokinase and glucose.


The structural changes that occur when an enzyme and a substrate join together bring specific parts of a substrate into alignment with specific parts of the enzyme’s active site. Amino acid side chains in or near the binding site can then act as acid or base catalysts, provide binding sites for the transfer of functional groups from one substrate to another or aid in the rearrangement of a substrate. The participating amino acids, which are usually widely separated in the primary sequence of the protein, are brought close together in the active site as a result of the folding and bending of the polypeptide chain or chains when the protein acquires its tertiary and quaternary structure. Binding to enzymes brings reactants close to each other and aligns them properly, which has the same effect as increasing the concentration of the reacting compounds.


Example 1

What type of interaction would occur between an OH group present on a substrate molecule and a functional group in the active site of an enzyme?Suggest an amino acid whose side chain might be in the active site of an enzyme and form the type of interaction you just identified.

Solution

An OH group would most likely engage in hydrogen bonding with an appropriate functional group present in the active site of an enzyme.Several amino acid side chains would be able to engage in hydrogen bonding with an OH group. One example would be asparagine, which has an amide functional group.

What type of interaction would occur between an COO− group present on a substrate molecule and a functional group in the active site of an enzyme?


Suggest an amino acid whose side chain might be in the active site of an enzyme and form the type of interaction you just identified.


One characteristic that distinguishes an enzyme from all other types of catalysts is its substrate specificity. An inorganic acid such as sulfuric acid can be used to increase the reaction rates of many different reactions, such as the hydrolysis of disaccharides, polysaccharides, lipids, and proteins, with complete impartiality. In contrast, enzymes are much more specific. Some enzymes act on a single substrate, while other enzymes act on any of a group of related molecules containing a similar functional group or chemical bond. Some enzymes even distinguish between D- and L-stereoisomers, binding one stereoisomer but not the other. Urease, for example, is an enzyme that catalyzes the hydrolysis of a single substrate—urea—but not the closely related compounds methyl urea, thiourea, or biuret. The enzyme carboxypeptidase, on the other hand, is far less specific. It catalyzes the removal of nearly any amino acid from the carboxyl end of any peptide or protein.


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Enzyme specificity results from the uniqueness of the active site in each different enzyme because of the identity, charge, and spatial orientation of the functional groups located there. It regulates cell chemistry so that the proper reactions occur in the proper place at the proper time. Clearly, it is crucial to the proper functioning of the living cell.


The lock-and-key model portrays an enzyme as conformationally rigid and able to bond only to substrates that exactly fit the active site. The induced fit model portrays the enzyme structure as more flexible and is complementary to the substrate only after the substrate is bound.


Urease has the greater specificity because it can bind only to a single substrate. Carboxypeptidase, on the other hand, can catalyze the removal of nearly any amino acid from the carboxyl end of a peptide or protein.


Key Takeaways

A substrate binds to a specific region on an enzyme known as the active site, where the substrate can be converted to product.The substrate binds to the enzyme primarily through hydrogen bonding and other electrostatic interactions.The induced-fit model says that an enzyme can undergo a conformational change when binding a substrate.Enzymes exhibit varying degrees of substrate specificity.

Exercises

1. What type of interaction would occur between each group present on a substrate molecule and a functional group of the active site in an enzyme?

a. COOH

b. NH3+

c. OH

d. CH(CH3)2

2. What type of interaction would occur between each group present on a substrate molecule and a functional group of the active site in an enzyme?

a. SH

b. NH2

c. C6H5

d. COO−

3. For each functional group in Exercise 1, suggest an amino acid whose side chain might be in the active site of an enzyme and form the type of interaction you identified.

4. For each functional group in Exercise 2, suggest an amino acid whose side chain might be in the active site of an enzyme and form the type of interaction you identified.


Answers

1.

a. hydrogen bonding

b. ionic bonding

c. hydrogen bonding

d. dispersion forces

3.

a. The amino acid has a polar side chain capable of engaging in hydrogen bonding; serine (answers will vary).

b. The amino acid has a negatively charged side chain; aspartic acid (answers will vary).

c. The amino acid has a polar side chain capable of engaging in hydrogen bonding; asparagine (answers will vary).

d. The amino acid has a nonpolar side chain; isoleucine (answers will vary).


Learning Objective

Describe how pH, temperature, and the concentration of an enzyme and its substrate influence enzyme activity.

The single most important property of enzymes is the ability to increase the rates of reactions occurring in living organisms, a property known as catalytic activity. Because most enzymes are proteins, their activity is affected by factors that disrupt protein structure, as well as by factors that affect catalysts in general. Factors that disrupt protein structure, as we saw in Section 9.1 "Proteins", include temperature and pH; factors that affect catalysts in general include reactant or substrate concentration and catalyst or enzyme concentration. The activity of an enzyme can be measured by monitoring either the rate at which a substrate disappears or the rate at which a product forms.


Concentration of Substrate

In the presence of a given amount of enzyme, the rate of an enzymatic reaction increases as the substrate concentration increases until a limiting rate is reached, after which further increase in the substrate concentration produces no significant change in the reaction rate (part (a) of Figure 9.12 "Concentration versus Reaction Rate"). At this point, so much substrate is present that essentially all of the enzyme active sites have substrate bound to them. In other words, the enzyme molecules are saturated with substrate. The excess substrate molecules cannot react until the substrate already bound to the enzymes has reacted and been released (or been released without reacting).


Figure 9.12 Concentration versus Reaction Rate

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(a) This graph shows the effect of substrate concentration on the rate of a reaction that is catalyzed by a fixed amount of enzyme. (b) This graph shows the effect of enzyme concentration on the reaction rate at a constant level of substrate.


Let’s consider an analogy. Ten taxis (enzyme molecules) are waiting at a taxi stand to take people (substrate) on a 10-minute trip to a concert hall, one passenger at a time. If only 5 people are present at the stand, the rate of their arrival at the concert hall is 5 people in 10 minutes. If the number of people at the stand is increased to 10, the rate increases to 10 arrivals in 10 minutes. With 20 people at the stand, the rate would still be 10 arrivals in 10 minutes. The taxis have been “saturated.” If the taxis could carry 2 or 3 passengers each, the same principle would apply. The rate would simply be higher (20 or 30 people in 10 minutes) before it leveled off.


Concentration of Enzyme

When the concentration of the enzyme is significantly lower than the concentration of the substrate (as when the number of taxis is far lower than the number of waiting passengers), the rate of an enzyme-catalyzed reaction is directly dependent on the enzyme concentration (part (b) of Figure 9.12 "Concentration versus Reaction Rate"). This is true for any catalyst; the reaction rate increases as the concentration of the catalyst is increased.


Temperature

A general rule of thumb for most chemical reactions is that a temperature rise of 10°C approximately doubles the reaction rate. To some extent, this rule holds for all enzymatic reactions. After a certain point, however, an increase in temperature causes a decrease in the reaction rate, due to denaturation of the protein structure and disruption of the active site (part (a) of Figure 9.13 "Temperature and pH versus Concentration"). For many proteins, denaturation occurs between 45°C and 55°C. Furthermore, even though an enzyme may appear to have a maximum reaction rate between 40°C and 50°C, most biochemical reactions are carried out at lower temperatures because enzymes are not stable at these higher temperatures and will denature after a few minutes.


Figure 9.13 Temperature and pH versus Concentration

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(a) This graph depicts the effect of temperature on the rate of a reaction that is catalyzed by a fixed amount of enzyme. (b) This graph depicts the effect of pH on the rate of a reaction that is catalyzed by a fixed amount of enzyme.


At 0°C and 100°C, the rate of enzyme-catalyzed reactions is nearly zero. This fact has several practical applications. We sterilize objects by placing them in boiling water, which denatures the enzymes of any bacteria that may be in or on them. We preserve our food by refrigerating or freezing it, which slows enzyme activity. When animals go into hibernation in winter, their body temperature drops, decreasing the rates of their metabolic processes to levels that can be maintained by the amount of energy stored in the fat reserves in the animals’ tissues.


Hydrogen Ion Concentration (pH)

Because most enzymes are proteins, they are sensitive to changes in the hydrogen ion concentration or pH. Enzymes may be denatured by extreme levels of hydrogen ions (whether high or low); any change in pH, even a small one, alters the degree of ionization of an enzyme’s acidic and basic side groups and the substrate components as well. Ionizable side groups located in the active site must have a certain charge for the enzyme to bind its substrate. Neutralization of even one of these charges alters an enzyme’s catalytic activity.

An enzyme exhibits maximum activity over the narrow pH range in which a molecule exists in its properly charged form. The median value of this pH range is called the optimum pH of the enzyme (part (b) of Figure 9.13 "Temperature and pH versus Concentration"). With the notable exception of gastric juice (the fluids secreted in the stomach), most body fluids have pH values between 6 and 8. Not surprisingly, most enzymes exhibit optimal activity in this pH range. However, a few enzymes have optimum pH values outside this range. For example, the optimum pH for pepsin, an enzyme that is active in the stomach, is 2.0.


The concentration of substrate X is low. What happens to the rate of the enzyme-catalyzed reaction if the concentration of X is doubled?


What effect does an increase in the enzyme concentration have on the rate of an enzyme-catalyzed reaction?


If the concentration of the substrate is low, increasing its concentration will increase the rate of the reaction.


An increase in the amount of enzyme will increase the rate of the reaction (provided sufficient substrate is present).


Key Takeaways

Initially, an increase in substrate concentration leads to an increase in the rate of an enzyme-catalyzed reaction. As the enzyme molecules become saturated with substrate, this increase in reaction rate levels off.The rate of an enzyme-catalyzed reaction increases with an increase in the concentration of an enzyme.At low temperatures, an increase in temperature increases the rate of an enzyme-catalyzed reaction. At higher temperatures, the protein is denatured, and the rate of the reaction dramatically decreases.An enzyme has an optimum pH range in which it exhibits maximum activity.

Exercises

1. In non-enzyme-catalyzed reactions, the reaction rate increases as the concentration of reactant is increased. In an enzyme-catalyzed reaction, the reaction rate initially increases as the substrate concentration is increased but then begins to level off, so that the increase in reaction rate becomes less and less as the substrate concentration increases. Explain this difference.

2. Why do enzymes become inactive at very high temperatures?

3. An enzyme has an optimum pH of 7.4. What is most likely to happen to the activity of the enzyme if the pH drops to 6.3? Explain.

4. An enzyme has an optimum pH of 7.2. What is most likely to happen to the activity of the enzyme if the pH increases to 8.5? Explain.


Answers

1. In an enzyme-catalyzed reaction, the substrate binds to the enzyme to form an enzyme-substrate complex. If more substrate is present than enzyme, all of the enzyme binding sites will have substrate bound, and further increases in substrate concentration cannot increase the rate.

3. The activity will decrease; a pH of 6.3 is more acidic than 7.4, and one or more key groups in the active site may bind a hydrogen ion, changing the charge on that group.


Learning Objectives

Explain what an enzyme inhibitor is.Distinguish between reversible and irreversible inhibitors.Distinguish between competitive and noncompetitive inhibitors.

In Section 9.4 "Enzyme Activity", we noted that enzymes are inactivated at high temperatures and by changes in pH. These are nonspecific factors that would inactivate any enzyme. The activity of enzymes can also be regulated by more specific inhibitors. Many compounds are poisons because they bind covalently to particular enzymes or kinds of enzymes and inactivate them (Table 9.4 "Poisons as Enzyme Inhibitors").


Table 9.4 Poisons as Enzyme Inhibitors

PoisonFormulaExample of Enzyme InhibitedAction
arsenateAsO43−glyceraldehyde 3-phosphate dehydrogenasesubstitutes for phosphate
iodoacetateICH2COO−triose phosphate dehydrogenasebinds to cysteine SH group
diisopropylfluoro-phosphate (DIFP; a nerve poison)
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acetylcholinesterasebinds to serine OH group

Irreversible Inhibition: Poisons

An irreversible inhibitor inactivates an enzyme by bonding covalently to a particular group at the active site. The inhibitor-enzyme bond is so strong that the inhibition cannot be reversed by the addition of excess substrate. The nerve gases, especially DIFP, irreversibly inhibit biological systems by forming an enzyme-inhibitor complex with a specific OH group of serine situated at the active sites of certain enzymes. The peptidases trypsin and chymotrypsin contain serine groups at the active site and are inhibited by DIFP.


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Reversible Inhibition

A reversible inhibitor inactivates an enzyme through noncovalent, more easily reversed, interactions. Unlike an irreversible inhibitor, a reversible inhibitor can dissociate from the enzyme. Reversible inhibitors include competitive inhibitors and noncompetitive inhibitors. (There are additional types of reversible inhibitors.) A competitive inhibitor is any compound that bears a structural resemblance to a particular substrate and thus competes with that substrate for binding at the active site of an enzyme. The inhibitor is not acted on by the enzyme but does prevent the substrate from approaching the active site.

The degree to which a competitive inhibitor interferes with an enzyme’s activity depends on the relative concentrations of the substrate and the inhibitor. If the inhibitor is present in relatively large quantities, it will initially block most of the active sites. But because the binding is reversible, some substrate molecules will eventually bind to the active site and be converted to product. Increasing the substrate concentration promotes displacement of the inhibitor from the active site. Competitive inhibition can be completely reversed by adding substrate so that it reaches a much higher concentration than that of the inhibitor.

Studies of competitive inhibition have provided helpful information about certain enzyme-substrate complexes and the interactions of specific groups at the active sites. As a result, pharmaceutical companies have synthesized drugs that competitively inhibit metabolic processes in bacteria and certain cancer cells. Many drugs are competitive inhibitors of specific enzymes.

A classic example of competitive inhibition is the effect of malonate on the enzyme activity of succinate dehydrogenase (Figure 9.14 "Competitive Inhibition"). Malonate and succinate are the anions of dicarboxylic acids and contain three and four carbon atoms, respectively. The malonate molecule binds to the active site because the spacing of its carboxyl groups is not greatly different from that of succinate. However, no catalytic reaction occurs because malonate does not have a CH2CH2 group to convert to CH=CH. This reaction will also be discussed in connection with the Krebs cycle and energy production. (For more information about the Krebs cycle, see Chapter 11 "Metabolic Pathways and Energy Production", Section 11.4 "Stage III of Catabolism".)


Figure 9.14 Competitive Inhibition

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(a) Succinate binds to the enzyme succinate dehydrogenase. A dehydrogenation reaction occurs, and the product—fumarate—is released from the enzyme. (b) Malonate also binds to the active site of succinate dehydrogenase. In this case, however, no subsequent reaction occurs while malonate remains bound to the enzyme.


To Your Health: Penicillin

Chemotherapy is the strategic use of chemicals (that is, drugs) to destroy infectious microorganisms or cancer cells without causing excessive damage to the other, healthy cells of the host. From bacteria to humans, the metabolic pathways of all living organisms are quite similar, so the search for safe and effective chemotherapeutic agents is a formidable task. Many well-established chemotherapeutic drugs function by inhibiting a critical enzyme in the cells of the invading organism.

An antibiotic is a compound that kills bacteria; it may come from a natural source such as molds or be synthesized with a structure analogous to a naturally occurring antibacterial compound. Antibiotics constitute no well-defined class of chemically related substances, but many of them work by effectively inhibiting a variety of enzymes essential to bacterial growth.

Penicillin, one of the most widely used antibiotics in the world, was fortuitously discovered by Alexander Fleming in 1928, when he noticed antibacterial properties in a mold growing on a bacterial culture plate. In 1938, Ernst Chain and Howard Florey began an intensive effort to isolate penicillin from the mold and study its properties. The large quantities of penicillin needed for this research became available through development of a corn-based nutrient medium that the mold loved and through the discovery of a higher-yielding strain of mold at a United States Department of Agriculture research center near Peoria, Illinois. Even so, it was not until 1944 that large quantities of penicillin were being produced and made available for the treatment of bacterial infections.

Penicillin functions by interfering with the synthesis of cell walls of reproducing bacteria. It does so by inhibiting an enzyme—transpeptidase—that catalyzes the last step in bacterial cell-wall biosynthesis. The defective walls cause bacterial cells to burst. Human cells are not affected because they have cell membranes, not cell walls.

Several naturally occurring penicillins have been isolated. They are distinguished by different R groups connected to a common structure: a four-member cyclic amide (called a lactam ring) fused to a five-member ring. The addition of appropriate organic compounds to the culture medium leads to the production of the different kinds of penicillin.

The penicillins are effective against gram-positive bacteria (bacteria capable of being stained by Gram’s stain) and a few gram-negative bacteria (including the intestinal bacterium Escherichia coli). They are effective in the treatment of diphtheria, gonorrhea, pneumonia, syphilis, many pus infections, and certain types of boils. Penicillin G was the earliest penicillin to be used on a wide scale. However, it cannot be administered orally because it is quite unstable; the acidic pH of the stomach converts it to an inactive derivative. The major oral penicillins—penicillin V, ampicillin, and amoxicillin—on the other hand, are acid stable.


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Some strains of bacteria become resistant to penicillin through a mutation that allows them to synthesize an enzyme—penicillinase—that breaks the antibiotic down (by cleavage of the amide linkage in the lactam ring). To combat these strains, scientists have synthesized penicillin analogs (such as methicillin) that are not inactivated by penicillinase.

Some people (perhaps 5% of the population) are allergic to penicillin and therefore must be treated with other antibiotics. Their allergic reaction can be so severe that a fatal coma may occur if penicillin is inadvertently administered to them. Fortunately, several other antibiotics have been discovered. Most, including aureomycin and streptomycin, are the products of microbial synthesis. Others, such as the semisynthetic penicillins and tetracyclines, are made by chemical modifications of antibiotics; and some, like chloramphenicol, are manufactured entirely by chemical synthesis. They are as effective as penicillin in destroying infectious microorganisms. Many of these antibiotics exert their effects by blocking protein synthesis in microorganisms.

Initially, antibiotics were considered miracle drugs, substantially reducing the number of deaths from blood poisoning, pneumonia, and other infectious diseases. Some seven decades ago, a person with a major infection almost always died. Today, such deaths are rare. Seven decades ago, pneumonia was a dreaded killer of people of all ages. Today, it kills only the very old or those ill from other causes. Antibiotics have indeed worked miracles in our time, but even miracle drugs have limitations. Not long after the drugs were first used, disease organisms began to develop strains resistant to them. In a race to stay ahead of resistant bacterial strains, scientists continue to seek new antibiotics. The penicillins have now been partially displaced by related compounds, such as the cephalosporins and vancomycin. Unfortunately, some strains of bacteria have already shown resistance to these antibiotics.


Some reversible inhibitors are noncompetitive. A noncompetitive inhibitor can combine with either the free enzyme or the enzyme-substrate complex because its binding site on the enzyme is distinct from the active site. Binding of this kind of inhibitor alters the three-dimensional conformation of the enzyme, changing the configuration of the active site with one of two results. Either the enzyme-substrate complex does not form at its normal rate, or, once formed, it does not yield products at the normal rate. Because the inhibitor does not structurally resemble the substrate, the addition of excess substrate does not reverse the inhibitory effect.

Feedback inhibition is a normal biochemical process that makes use of noncompetitive inhibitors to control some enzymatic activity. In this process, the final product inhibits the enzyme that catalyzes the first step in a series of reactions. Feedback inhibition is used to regulate the synthesis of many amino acids. For example, bacteria synthesize isoleucine from threonine in a series of five enzyme-catalyzed steps. As the concentration of isoleucine increases, some of it binds as a noncompetitive inhibitor to the first enzyme of the series (threonine deaminase), thus bringing about a decrease in the amount of isoleucine being formed (Figure 9.15 "Feedback Inhibition of Threonine Deaminase by Isoleucine").


Figure 9.15 Feedback Inhibition of Threonine Deaminase by Isoleucine

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Threonine deaminase is the first enzyme in the conversion of threonine to isoleucine. Isoleucine inhibits threonine deaminase through feedback inhibition .


A competitive inhibitor structurally resembles the substrate for a given enzyme and competes with the substrate for binding at the active site of the enzyme. A noncompetitive inhibitor binds at a site distinct from the active site and can bind to either the free enzyme or the enzyme-substrate complex.


Key Takeaways

An irreversible inhibitor inactivates an enzyme by bonding covalently to a particular group at the active site.A reversible inhibitor inactivates an enzyme through noncovalent, reversible interactions.A competitive inhibitor competes with the substrate for binding at the active site of the enzyme.A noncompetitive inhibitor binds at a site distinct from the active site.

See more: How Many Valance Electrons Does Oxygen Have ? How Many Valence Electrons Does Oxygen Have


What amino acid is present in the active site of all enzymes that are irreversibly inhibited by nerve gases such as DIFP?


Oxaloacetate (OOCCH2COCOO) inhibits succinate dehydrogenase. Would you expect oxaloacetate to be a competitive or noncompetitive inhibitor? Explain.