Enzymes

Question 1

THE NATURE OF ENZYMES

Answer 1

Enzymes are catalysts that greatly increase the rate of chemical reactions and thus make possible the numerous and diverse metabolic processes that occur in the human body. Catalysts increase the rate of a reaction without affecting its equilibrium.

Enzymes can increase the rate of physiological reactions by as much as 10”‘-fold.

They accomplish this feat by decreasing the amount of energy required for activation of the initial reactants (substrates), thereby increasing the percentage of substrate molecules that have sufficient energy to react.

With the exception of a few ribonucleic acid (RNA) molecules (ribozymes) that
catalyze reactions involving nucleic acids, enzymes are proteins.

Every enzyme has an active site that is composed of specific amino acid side chains which are brought into close proximity when the enzyme is folded into its active conformation. During the course of the reaction that it catalyzes, the enzyme’s active site stabilizes the transition state, which is an intermediate conformation between substrates and products.

The interaction between active site and substrate(s) is thus responsible for the catalytic efficiency of the enzyme as well as its substrate specificity. After the reaction occurs, the products are released from the enzyme and the active site is available to bind additional substrate molecules.

Question 2

TYPES OF ENZYMES

Answer 2

1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
7. Nonenzymatic Reactions

Question 3

TYPES OF ENZYMES
Oxidoreductases

Answer 3

Oxidative reactions remove electrons, usually one or two electrons per molecule
of substrate, while reductive reactions accomplish the converse. The substrate in
an oxidation-reduction reaction may be a metal, as in the case of the one-electron
oxidation of the ferrous ion of hemoglobin to the ferric ion of methemoglobin, or an organic compound as illustrated by the two-electron, reversible oxidation of lactate
to pyruvate.

Oxidoreductases transfer electrons from one compound to another, thus changing the oxidation state of both substrates.

Some oxidoreductases, such as lactate dehydrogenase, catalyze the removal of two hydrogen atoms (two electrons plus two hydrogen ions) to an acceptor molecule such as nicotinamide-adenine dinucleotide (NADf) as illustrated by the lactate dehydrogenase reaction:
lactate + NAD+ <-> pyruvate + NADH + H’

A second cofactor that serves as an acceptor of hydrogen atoms is flavin-adenine dinucleotide (FAD):
succinate + FAD -> fumarate + FADH2

In general, most oxidation-reduction (redox) reactions that oxidize oxygen-bearing carbons utilize NAD+ (or the related cofactor NADP+), whereas reductions or oxidations of carbon atoms that do not have oxygen attached utilize flavin mononucleotides (FMN or FAD).

Other oxidoreductases, such as 5-lipoxygenase, are dioxygenases,
which catalyze the addition of both atoms of molecular oxygen into the substrate:

arachidonic acid + 02 -> 5-hydroperoxyeicosatetraenoic acid

Still other oxidoreductases are monooxygenases or mixed-function oxidases, which catalyze even more complex reactions. For example, phenylalanine hydroxylase catalyzes the reaction

phenylalanine + 02
+ BH4 -> tyrosine + BH2 + H2O

In this reaction, two organic substrates are oxidized: One atom of molecular oxygen is used to oxidize phenylalanine; the other combines with the two hydrogen atoms removed from tetrahydrobiopterin (BH4), generating dihydrobiopterin (BH2) and
water.

Question 4

TYPES OF ENZYMES
Transferases

Answer 4

Transferases catalyze reactions in which a functional group is transferred from one
compound to another.

Transaminases, such as aspartate aminotransferase, catalyze the reversible transfer of an amino group from an amino acid to an alpha-ketoacid, thus generating a new amino acid and a new alpha-ketoacid:

aspartate + alpha-ketoglutarate <-> oxaloacetate + glutamate

Similarly, kinases transfer phosphate groups from adenosine triphosphate (ATP) to acceptor molecules such as glucose in the reaction catalyzed by hexokinase or glucokinase:

glucose + ATP + glucose 6-phosphate + adenosine diphosphate (ADP)

Unlike the aminotransferase reactions, which are reversible, most reactions catalyzed by kinases are irreversible under physiological conditions.

Question 5

TYPES OF ENZYMES
Hydrolases

Answer 5

Hydrolases cleave carbon-oxygen, carbon-nitrogen, or carbon-sulfur bonds by adding water across the bond.

One example of a hydrolase is the digestive enzyme maltase, which hydrolyzes the glycosidic bond in the disaccharide maltose:
maltose + H20 -> 2 glucose

Question 6

TYPES OF ENZYMES
Lyases

Answer 6

**Lyases cleave carbon-oxygen, carbon-nitrogen, or carbon-sulfur bonds but do so without addition of water and without oxidizing or reducing the substrates. **

A good example of a lyase is aldolase A, which as an enzyme of the glyco-
lytic pathway, catalyzes the reversible cleavage of the six-carbon sugar
fructose 1,6-bisphosphate into two three-carbon sugar phosphates:

fructose 1,6-bisphosphate <-> glyceraldehyde 3-phosphate
+ dihydroxyacetone phosphate

Note that in the reverse reaction, aldolase A functions as a synthase, forming a new
C-C bond.

Question 7

TYPES OF ENZYMES
Isomerases

Answer 7

Isomerases catalyze intramolecular rearrangements of functional groups that reversibly interconvert optical or geometric isomers.

One example is glucose 6-phosphate isomerase (Fig. 2-8A), which converts glucose 6-phosphate, an aldo-
sugar phosphate, to the isomeric keto-sugar phosphate, fructose 6-phosphate:

glucose 6-phosphate <-> fructose 6-phosphate

When an isomerase catalyzes an intramolecular rearrangement involving movement of a functional group, it is called a mutuse.

For example, as part of the two metabolic pathways that synthesize and break down glycogen, phosphoglucomutase (Fig. 2-8B)
catalyzes the reversible transfer of a phosphate group between the hydroxyl group on C1 (of the hemiacetyl ring form of glucose) and the C6 hydroxyl group of glucose:

glucose 6-phosphate <-> glucose 1 -phosphate

Question 8

TYPES OF ENZYMES
Ligases

Answer 8

**Ligases catalyze biosynthetic reactions that form a covalent bond between two substrates. **

An example of a ligase is pyruvate carboxylase, which forms a new C-C bond by adding C02 from bicarbonate to pyruvate, the three-carbon end product of aerobic glycolysis

pyruvate + HCO3- + ATP -> oxaloacetate + ADP + Pi

Some ligases that catalyze synthetic reactions in which two substrates are joined and a nucleotide triphosphate (e.g., ATP) is hydrolyzed are designated by the term synthetase.

In contrast, the term synthase is used to describe enzymes that catalyze reactions in which two substrates come together to form a product, but a nucleotide triphosphate is not involved in the reaction.

An example of a synthase is citrate synthase, where the energy to drive the reaction is provided by the thioester of acetyl-CoA:

oxaloacetate + acetyl-CoA -> citrate + CoASH

Question 9

TYPES OF ENZYMES
Nonenzymatic Reactions

Answer 9

Not all physiologically or pathophysiologically relevant reactions that take place in the body are catalyzed by enzymes.

For example, the covalent attachment of glucose to hemoglobin to form glycated hemoglobin (HbAlc) occurs spontaneously and does not involve an enzyme.

The fact that the extent of this glycation reaction in blood is determined solely by the plasma glucose concentration is the basis for the usefulness of the HbAlc measurement as a way to monitor glucose control. The high reactivity of
glucose (as well as galactose and other monosaccharides) with proteins is attributable to the intrinsic affinity of aldehyde groups for the amino groups of proteins, resulting in protein adducts that can act as neoantigens.

Similarly, the covalent attachment of
acetaldehyde, an intermediate in ethanol metabolism, to a wide range of proteins may account for some of the pathology associated with excessive consumption of ethanol.

Another example of an important nonenzymatic reaction in humans is the autooxidation of oxyhemoglobin to methemoglobin, which generates the superoxide anion:

hemoglobin (Fe2+) + O2 -> methemoglobin (Fe3+) + O2;

Methemoglobin does not bind oxygen and is a potent oxidizing agent that can damage the red cell membrane.

Question 10

SMALL MOLECULES AND METAL IONS CAN CONTRIBUTE
TO ENZYME-BASED CATALYSIS

Answer 10

1. Cofactors
2. Vitamins Are Components of Many Enzymatic Cofactors or Coenzymes
   Thiamine (Vitamin B7)
   Riboflavin (Vitamin B2)
   Niacin (Vitamin B3)
   Pyridoxine, Pyridoxal, and Pyridoxamine
   Biotin
   Folate
   Cobalamin (Vitamin B12)
   Pantothenic Acid
   Ascorbic Acid (Vitamin C)
   Vitamin K.
   Not All Cofactors Are Derived from Vitamins

Question 11

SMALL MOLECULES AND METAL IONS CAN CONTRIBUTE TO ENZYME-BASED CATALYSIS
Cofactors

Answer 11

Enzymatic catalysis often involves utilization of an additional small organic molecule called a cofactor.

Certain cofactors, such as biotin and thiamine pyrophosphate, function only when they are attached covalently to their respective enzymes. In such cases the enzyme-coenzyme complex is called a holoenzyme, whereas the term apoenzyme refers to the protein component alone.

In other cases, the cofactor acts more like a second substrate. A good example of this is NAD+, which is converted to
NADH + H when it receives two hydrogen atoms (or two electrons plus protons) during the course of the redox reaction catalyzed by lactate dehydrogenase. The (e.g., NADH molecule subsequently transfers the hydrogen atoms to another acceptor FAD in the mitochondrial electron transport chain) and is thus available to participate in the catalytic dehydrogenation of another molecule of lactate. These NAD+-utilizing enzymes are usually designated as dehydrogenases.

Most cofactors usually participate in the catalysis of many different reactions, often
using a similar reaction mechanism. The cofactor does this by binding to the various enzymes, each of which has a particular active site whose structure and binding properties determine its unique substrate specificity.

Thus, lactate dehydrogenase catalyzes the reaction
lactate + NAD+ <-> pyruvate + NADH + H+

whereas alcohol dehydrogenase catalyzes the reaction
ethanol + NAD+ <-> acetaldehyde + NADH + H+

Question 12

SMALL MOLECULES AND METAL IONS CAN CONTRIBUTE TO ENZYME-BASED CATALYSIS
Vitamins Are Components of Many Enzymatic Cofactors or Coenzymes

Answer 12

Vitamins are small organic molecules that are not synthesized in the body and are
therefore essential dietary nutrients.

Many of the vitamins are cofactors or components of cofactors. Because they play a catalytic role, they are required in the diet in only small amounts and are referred to as rnicronutrients. The vitamins that are cofactors or cofactor precursors include all the water-soluble B vitamins, vitamin C, and the fat-soluble vitamin K

Thiamine (Vitamin B7)
Riboflavin (Vitamin B2)
Niacin (Vitamin B3)
Pyridoxine, Pyridoxal, and Pyridoxamine
Biotin
Folate
Cobalamin (Vitamin B12)
Pantothenic Acid
Ascorbic Acid (Vitamin C)
Vitamin K.
Not All Cofactors Are Derived from Vitamins

Question 13

Thiamine (Vitamin B7)

Answer 13

Thiamine is utilized to synthesize thiamine pyrophosphate, which contributes to the transfer of active aldehyde intermediates during several reactions of carbohydrate metabolism aka. pyruvate dehydrogenase, the tricarboxylic acid cycle enzyme a-ketoglutarate dehydrogenase and transketolase, an enzyme that is a component of the pentose phosphate
pathway.

Question 14

Riboflavin (Vitamin B2).

Answer 14

Riboflavin is a component of FAD (flavin-
adenine dinucleotide) and FMN (flavin mononucleotide), which participate
in numerous oxidation-reduction (redox) reactions and the process of ATP generation in mitochondria.

FAD-linked dehydrogenases convert succinate to fumarate in the TCA cycle and fatty acyl-CoA to P-hydroxy fatty acyl-CoA during P-oxidation of fatty acids.

Question 15

Niacin (Vitamin B3)

Answer 15

Niacin is a component of NAD+ (nicotinamide- adenine dinucleotide), and NADP+ (nicotinamide-adenine dinucleotide phosphate), which participate in many redox reactions, such as those catalyzed by lactate dehydrogenase and fatty acyl-CoA dehydrogenase.

NADP+ differs from NAD+ in that it has a phosphate group on C6 of the ribose moiety to which the adenosine moiety is attached.

NADH, the reduced form of NAD+, also donates electrons to the mitochondrial electron transport chain, which is a series of oxidation-reduction reactions that ultimately generate ATP.

NADP+ is a substrate or cofactor in the
glucose 6-phosphate dehydrogenase reaction of the pentose phosphate pathway, and NADPH provides reducing equivalents for the synthesis of fatty acids and cholesterol.

Question 16

Pyridoxine, Pyridoxal, and Pyridoxamine

Answer 16

These are forms of vitamin B6 and precursors of pyridoxal phosphate (PLP).

PLP is a cofactor for many enzymes that catalyze reactions involving amino acids, such as the various aminotransferases, amino acid decarboxylases, and the ligase enzyme delta-amino- levulinic acid (ALA) synthetase, which catalyzes the regulated step of heme synthesis.

Question 17

Biotin

Answer 17

Biotin is active when it is attached covalently to enzymes. It binds C02 and transfers this one-carbon unit to organic acceptors (e.g., acetyl-CoA, pyruvate) as part of the catalytic mechanism of enzymes such as acetyl-CoA
carboxylase and pyruvate carboxylase.

Question 18

Folate

Answer 18

Folate is the precursor of tetrahydrofolate (THF), which is the cofactor involved in the transfer of one-carbon groups other than C02. THF plays a central role in the synthesis of purines, which are the building blocks for both
deoxyribonucleic acid (DNA) and RNA.

Question 19

Cobalamin (Vitamin B12).

Answer 19

Cobalamin is the cofactor that participates
in the transfer of a methyl group in the regeneration of methionine from homo-
cysteine. Cobalamin is also the precursor of deoxyadenosylcobalamin, which is the
cofactor for methylmalonyl-CoA mutase, an enzyme involved in the metabolism of
propionic acid.

Question 20

Pantothenic Acid

Answer 20

Pantothenic acid is a component of coenzyme A (CoASH) and acyl carrier protein (ACP). The sulfhydryl group of CoASH forms thioester bonds with the carboxyl groups of acetate, long-chain fatty acids, and other organic acids. CoASH serves as a carrier for the activated forms of organic acids during many reactions, including those involved in the TCA cycle, fatty acid oxidation, the catabolism of the carbon skeletons of branched-chain amino acids, and the conjugation of bile salts with glycine or taurine. Acyl carrier protein is the carrier of acyl groups during the de novo synthesis of fatty acids.

Question 21

Ascorbic Acid (Vitamin C)

Answer 21

Ascorbic acid is a cofactor in hydroxyl-
ation reactions, most prominently the hydroxylation of proline residues of collagen (Fig. 2-10) and the synthesis of norepinephrine from dopamine. Ascorbate is oxidized to dehydroascorbate during the course of these hydroxylation reactions and is
regenerated by dehydroascorbate reductase, using reduced glutathione (GSH) as the source of reducing equivalents and generating oxidized glutathione (GSSG):
dehydroascorboate + 2GSH -+ ascorbic acid + GSSG

Question 22

Vitamin K.

Answer 22

The two major dietary molecules with vitamin K activity are menaquinone, synthesized by bacteria, and phylloyuinone, a product of green plants.
Vitamin K is the cofactor for enzymes that y -carboxylate specific glutamate residues
of calcium-binding proteins (Fig. 2- 1 I), such as prothrombin and other proteins of the blood-clotting cascade, and osteocalcin, a major bone protein. Vitamin K undergoes oxidation during y -carboxylation reactions and is subsequently regenerated in two reduction reactions catalyzed by vitamin K epoxide reductase and vitamin K
reductase, respectively.

Question 23

SMALL MOLECULES AND METAL IONS CAN CONTRIBUTE TO ENZYME-BASED CATALYSIS
Not All Cofactors Are Derived from Vitamins

Answer 23

It is worth emphasizing that not all cofactors are synthesized from a vitamin. For example, since tetrahydrobiopterin (BH4, Fig. 2-4B), the cofactor for phenylalanine hydroxylase and
other enzymes that hydroxylate aromatic amino acids, is synthesized in the body
from guanosine triphosphate (GTP), it is not a vitamin. Similarly, lipoic acid, which
is one of several cofactors for the pyruvate dehydrogenase and a-ketoglutarate dehydrogenase complexes, is not a vitamin. It should also be noted that not all vitamins are precursors of cofactors. Indeed, vitamin K is the only one of the four fat-soluble vitamins that plays a direct catalytic role in an enzyme-catalyzed reaction in the body.
Two other fat-soluble vitamins, retinol (vitamin A) and cholecalciferol (vitamin D),
are actually precursors of hormones that regulate transcription of DNA, and thus
gene expression. Retinol is also the precursor of 11-&-retinal, which is an important constituent of rhodopsin, the visual pigment of the eye. a-Tocopherol (vitamin E), the fourth fat-soluble vitamin, is an antioxidant.

Question 24

Many Enzymes Utilize Metal Ions as Part of
Their Catalytic Mechanisms

Answer 24

Many enzymes utilize inorganic ions to bind the substrate and polarize critical func-
tional groups. Examples of metal ions and the enzymes they function with include:
Zn2+: alcohol dehydrogenase, carboxypeptidase
Mg2+: ATP-dependent reactions such as hexokinase
Fe3+ and Cuz+: components of the cytochrome oxidase complex, which catalyzes the last step in the electron transport chain in which the protons and electrons are transferred to molecular oxygen
Se2+: glutathione peroxidase, which is involved in the cellular defense against free radicals

Question 25

HOW DO ENZYMES WORK?

Answer 25

Biological catalysts increase the rate of a chemical reaction, permitting reactions to
occur that would otherwise be so slow as to be incompatible with life. Mammalian
enzymes have evolved to catalyze reactions under physiological conditions, that is, at 37°C and usually at a pH near neutrality. They commonly accelerate reactions by factors of lo6 to 10” and are usually highly specific for their substrates.

The active site of an enzyme is the pocket in the protein where the substrate
or substrates are bound. Substrates are bound to enzymes in what is referred to an enzyme-substrate (ES) complex by multiple weak (usually noncovalent) interactions, particularly ionic and hydrogen bonds. Binding of substrates to the enzyme’s active site stabilizes the reaction intermediate or transition state, thereby decreasing the amount of activation energy required for the reaction to occur (Fig. 2- 1).

Theoretically, all chemical reactions are reversible to some extent. Enzymes catalyze both the forward and reverse reactions.

What Determines the Direction in Which Reversible
Reactions Proceed?
Irreversible Reactions
Isozymes Are Different Proteins That Catalyze
the Same Reaction

Question 26

HOW DO ENZYMES WORK?
What Determines the Direction in Which Reversible Reactions Proceed?

Answer 26

An example of a reversible reaction is the one catalyzed by lactate dehydrogenase:
lactate + NAD’ + pyruvate + NADH + H’
Whether one starts with the substrates (shown on the left) or the products (on the right), the lactate dehydrogenase reaction, like all reactions, will eventually reach an equilibrium or steady-state condition. At equilibrium, the relative proportion of reactants on the left and products on the right will be determined by the change in free energy of the reaction (AGO’); in other words, the reaction will proceed in the
direction that releases energy (AGO’ < 0) rather than one that requires a net input of energy.
For reversible reactions, the major factor that determines the rates of reactions
in the forward and reverse directions is the relative concentration of substrates and products. For reactions like the one catalyzed by lactate dehydrogenase, the direction of the reaction is determined primarily by the NADH/NAD+ ratio. Thus, when exercising muscle produces more NADH than can be utilized by the mitochondria1 oxidative phosphorylation system, the buildup of NADH drives lactate dehydrogenase to produce lactate from pyruvate. Conversely, when hepatocytes are actively utilizing
NADH for ATP production via oxidative phosphorylation, the NADH level falls
and the concentration of NAD+ increases, thereby causing lactate dehydrogenase to
generate pyruvate from lactate.

Question 27

HOW DO ENZYMES WORK?
Irreversible Reactions

Answer 27

There are many reactions that are essentially irreversible under physiological conditions. These irreversible reactions are exergonic, meaning that they give off significant energy. Biochemists consider a reaction to be irreversible when the free-
energy change (AGO’) is -4 kcal/mol or more negative. An example of a physiologically irreversible reaction is that catalyzed by glucose 6-phosphatase:

glucose 6-phosphate + H2O -> glucose + Pi

The reverse reaction, that is, the formation of glucose 6-phosphate, would require the input of significant energy. Neither glucokinase nor hexokinase, the two enzymes that catalyze the synthesis of glucose 6-phosphate from free glucose, can directly reverse the reaction catalyzed by glucose 6-phosphatase. Instead, both of these enzymes utilize the energy associated with one of the high-energy bonds of ATP to phosphorylate
glucose:

glucose + ATP -+ glucose 6-phosphate + ADP

Acetyl-CoA carboxylase is another enzyme that utilizes the high-energy y -phosphate
bond of ATP to drive a reaction that would be irreversible without the participation
of ATP:

acetyl-CoA + CO2 + ATP + H20 -> malonyl-CoA + ADP + Pi

In this case, the terminal (y) phosphate of ATP (Fig. 1-2) is not incorporated into
the product of the reaction. Instead, two reactions (hydrolysis of ATP and carboxyl-
ation of acetyl-CoA) are coupled, with the favorable (energy-yielding or extqonic)
hydrolysis of ATP being used to drive the unfavorable (energy-requiring or ender-
gonic) carboxylation of acetyl-CoA.

Question 28

HOW DO ENZYMES WORK?
Isozymes Are Different Proteins That Catalyze the Same Reaction

Answer 28

As described above, glucokinase and hexokinase both catalyze the synthesis of glucose 6-phosphate from glucose and ATP. However, the two enzymes differ with regard to both their catalytic properties and their protein structures, and are therefore called isozymes or isoenzymes. Hexokinase, the isozyme present in almost every cell of the body, has a high affinity for glucose and is therefore active even at relatively low concentrations of glucose (Fig. 2- 12). By contrast, glucokinase, which
is found primarily in liver, is relatively inactive at low concentrations of glucose.
Glucokinase has a higher maximal activity than hexokinase and is able to respond to
increased blood glucose concentrations by rapidly synthesizing glucose 6-phosphate.
Biochemists quantify these differences by indicating that glucokinase has both a
higher Vmax (maximal reaction velocity) and a higher Km (the substrate concentration required to support half-maximal activity) than hexokinase. As shown in Figure 2- 12, the K, of hexokinase for glucose is 0.01 mM, while the lower affinity of glucokinase
for glucose is reflected by its higher K, of 5 to 10 mM. This difference in K,, values
between the two isozymes permits the liver to remove glucose rapidly from the blood when the glucose concentration is high, while leaving glucose available for glucose- dependent tissues (e.g., red blood cells, brain) when the body’s glucose reserves are low.

Question 29

HOW IS ENZYME ACTIVITY REGULATED?

Answer 29

Regulation of the functioning of the many enzymes in the body is central to co-
ordinating the multiple pathways of metabolism and maintaining homeostasis. One key mechanism for regulating the level of activity of a particular enzyme in a cell is regulation of gene expression, since if an enzyme is not synthesized in the appropriate cell or at a particular time, the reaction it catalyzes will not occur.
The activities of existing enzymes are themselves also regulated, both by intracellular availability of metabolites and by covalent modifications (e.g., phosphorylation).
In addition, many pharmaceutical agents act by inhibiting the activity of one or more enzymes. For example, patients who have elevated plasma cholesterol levels may be prescribed one of the statins, a class of drugs that inhibit HMG-CoA reductase, which catalyzes the rate-limiting step in cholesterol synthesis. The major mechanisms for regulation of enzymatic activity are described below.

Competitive Enzyme Inhibition
Noncompetitive Enzyme Inhibition
Allosteric Regulation
Regulation of Enzyme Activity by Covalent Modification
Induction of Enzyme Synthesis

Question 30

HOW IS ENZYME ACTIVITY REGULATED?
Competitive Enzyme Inhibition

Answer 30

Competitive inhibition occurs when a molecule that is not a substrate for the enzyme in question, but which is structurally similar to the substrate, competes with the substrate and blocks its binding to the active site of the enzyme.
Occupation of the active site by the inhibitor decreases the activity of the enzyme, particularly when the concentration of the substrate is low relative to that of the inhibitor.

Many pharmaceutical agents are competitive inhibitors of specific enzymes. For example, dicumarol inhibits catalysis involving vitamin K. Since many enzymes in the blood-clotting cascade are activated by y -carboxylation, dicumarol acts as an
anticoagulant that reduces the risk of thrombus formation.
In some cases, two different molecules may both be substrates for the same
enzyme, with each acting as a competitive inhibitor of the metabolism of the other.
One such example is alcohol dehydrogenase, which catalyzes the oxidation of both ethanol and methanol:
ethanol + NAD+ -> acetaldehyde + NADH + H+
methanol + NAD+ -> formaldehyde + NADH + H+
Although methanol itself is intoxicating, it is the metabolites of methanol (formalde-
hyde and formic acid) that are responsible for the blindness and death that result
from methanol poisoning. One treatment for acute methanol poisoning involves in-
travenous administration of ethanol (plus glucose). Ethanol acts as a competitive
inhibitor of the conversion of methanol to formaldehyde, thereby preventing ac-
cumulation of toxic metabolites until the methanol can be cleared by the kidneys.
Glucose is administered to correct the hypoglycemia caused by ethanol.

Question 31

HOW IS ENZYME ACTIVITY REGULATED?
Noncompetitive Enzyme Inhibition

Answer 31

A noncompetitive inhibitor binds to its target enzyme and cannot be displaced by excess substrate. Thus, this type of inhibitor diminishes the fraction of the enzyme pool that is catalytically competent.

Aspirin’s action as a noncompetitive inhibitor is the major reason that it is a
drug of choice for long-term therapy to decrease the risk of cardiovascular crises.
Aspirin is a member of a class of drugs called nonsteroidal anti-inflammatory agents (NSAIDs) that inhibit the cyclooxygenase isozymes, thereby decreasing thromboxane
production and platelet aggregation. Aspirin is an irreversible inhibitor since the molecule covalently acetylates a serine residue at the active site of the enzyme, inactivating cyclooxygenase permanently. By contrast, the inhibitory actions of other NSAIDs, such as ibuprofen, are attributable to reversible, noncovalent interactions between drug and enzyme. The reaction of aspirin with cyclooxygenase is particularly
effective in platelets because platelets are incapable of synthesizing new enzyme
protein.
Some of the deleterious effects of heavy metals, such as mercury and lead, re-
sult from their actions as noncompetitive enzyme inhibitors. For example, mercury
inhibits glyceraldehyde 3-phosphate dehydrogenase, an enzyme in the glycolytic pathway, while lead inhibits heme synthesis.

Question 32

HOW IS ENZY
ME ACTIVITY REGULATED?
Allosteric Regulation

Answer 32

Allosteric regulation, as this phenomenon is called, provides a mechanism
by which enzymatic activities can be modulated by compounds that have little or no structural similarity to the substrate(s) but which instead, reflect the overall metabolic state or needs of the cell. Allosteric enzymes usually exhibit sigmoidal (S-shaped) kinetic curves rather than simple hyperbolic curves. Activators
of allosteric enzymes shift the V vs. S curve to the left, whereas allosteric inhibitors shift the curve to the right.

End-Product Inhibition.
Regulation by Molecules That Signal the Availability of Precursors.
Regulation by the Energy Charge of the Cell

Question 33

HOW IS ENZYME ACTIVITY REGULATED?
Allosteric Regulation

Answer 33

End-Product Inhibition.

There are many instances in which the final endproduct of a multienzyme metabolic pathway is an allosteric inhibitor of an enzyme that catalyzes an early and irreversible step of the pathway. This form of allosteric regulation prevents accumulation of additional end product and of metabolic intermediates once a cell has sufficient supplies of that metabolic end product. Examples of this are seen in the pathways that generate heme, long-chain fatty acids, and cholesterol, where the end products inhibit delta-aminolevulinic acid synthase, acetyl-CoA carboxylase, and HMG-CoA reductase, respectively.

Regulation by Molecules That Signal the Availability of Precursors.

Allosteric regulation provides a mechanism by which flux through a particular pathway can be rendered responsive to the overall nutritional state and needs of the cell. One such important small regulatory metabolite is citrate, an intermediate in the tricarboxylic acid cycle. Citrate allosterically stimulates liver cells to synthesize both fatty acids and glucose (gluconeogenesis) while inhibiting the breakdown of glucose by glycolysis.

Regulation by the Energy Charge of the Cell.

Allosteric mechanisms also serve to regulate many metabolic pathways in response to a high ATP/ADP
ratio, which is indicative of a plentiful supply of energy, or conversely, to high concentrations of ADP and adenosine 5’-monophosphate (AMP), which occur when ATP supplies have been depleted. An enzyme whose activity is regulated by the energy charge of the cell is the muscle isozyme of glycogen phosphorylase, which releases glucose (as glucose 1-phosphate) from glycogen stores. AMP is an allosteric activator of glycogen phosphorylase, and ATP is an allosteric inhibitor of the enzyme.

Question 34

HOW IS ENZYME ACTIVITY REGULATED?Regulation of Enzyme Activity by Covalent Modification

Answer 34

1. Phosphorylation/Dephosphorylation.
2. Hydrolytic Cleavage of Inhibitory Peptides
   3.

Question 35

HOW IS ENZYME ACTIVITY REGULATED?Regulation of Enzyme Activity by Covalent Modification
Phosphorylation/Dephosphorylation

Answer 35

The most common covalent
modification utilized in regulating human metabolism is the reversible phosphoryl-
ation of enzyme proteins. In most cases, phosphorylation is the result of a hormone- stimulated signal-transduction cascade, thus providing a mechanism by which intracellular enzymatic activity can be modulated in response to intercellular signaling.
For example, glucagon and epinephrine both stimulate the activity of a
serine-threonine protein kinase called protein kinase A (PKA). Briefly, this particu-
lar signaling pathway involves binding of glucagon or epinephrine to its respective
transmembrane receptors, activation of a GTP-binding or G-protein, and activation
of adenylyl cyclase, an enzyme that synthesizes cyclic AMP (CAMP) from ATP

ATP -> CAMP + PPi

CAMP is an allosteric activator of PKA. Once activated, PKA uses ATP to phos-
phorylate specific serine and threonine residues on critical enzymes that regulate
the flux of intermediates through key metabolic pathways. As discussed in more
detail in Chapter 8, PKA-catalyzed phosphorylation activates enzymes involved with glycogen breakdown (glycogen phosphorylase).
PKA-catalyzed phosphorylation also
activates enzymes involved with mobilization of triacylglycerol stores and gluconeogenesis. Concurrently and conversely, PKA-catalyzed protein phosphorylation inhibits enzymes involved in glycogen and fatty acid metabolism. The simultaneous
phosphorylation of multiple enzymes provides a coordinated response to the body’s need to mobilize endogenous fuels during fasting or in response to stress.

Protein phosphorylation catalyzed by PKA can be reversed. Among the many
effects of stimulation of cells by insulin is the activation of a signal-transduction
cascade that results ultimately in the activation of protein phosphatase- 1. Protein phosphatase-1 hydrolyzes the phosphate moieties from phosphoserine and phospho- threonine residues of many enzymes, thereby reversing the activation or inactivation that occurred when those enzymes were phosphorylated. Accordingly, insulin reverses the metabolic effects of glucagon and epinephrine, and switches the direction
of key metabolic processes to meet the body’s needs in the fed state when there is active synthesis of triacylglycerol in adipocytes and hepatocytes as well as synthesis and storage of glycogen in muscle and liver.

Question 36

HOW IS ENZYME ACTIVITY REGULATED?Regulation of Enzyme Activity by Covalent Modification
Hydrolytic Cleavage of Inhibitory Peptides.

Answer 36

A number of enzymes, particularly digestive enzymes synthesized in the pancreas and the liver-synthesized
proteases of the blood-clotting cascade, are secreted from their sites of synthesis in an inactive or zymogen form. Activation requires proteolytic hydrolysis of the pro-
enzyme and release of a polypeptide, which then permits the remaining polypeptide fragment to alter its three-dimensional structure to one in which its active site and associated substrate binding pocket are configured correctly for catalysis. Thus, the digestive enzyme trypsin is secreted from the pancreas in the form of an inactive precursor, trypsinogen. Once in the lumen of the small intestine, a brush-border
protease called enteropeptidase hydrolyzes one peptide bond within the trypsinogen molecule, thereby releasing the inhibitory peptide and generating active trypsin.
Secretion of trypsin in its zymogen form limits its activity to the digestive tract, thus
protecting the pancreas and pancreatic duct from proteolytic damage.

Question 37

HOW IS ENZYME ACTIVITY REGULATED
Induction of Enzyme Synthesis

Answer 37

Hormonal regulation of enzymatic activity can also occur through stimulation or
inhibition of transcription of genes that encode key metabolic enzymes.

Hydrocortisone, a glucocorticoid hormone synthesized by the adrenal cortex, acts by entering the cell and binding to certain proteins in the cytosol that serve as glucocorticoid receptors. The hydrocortisone-glucocorticoid receptor complex then translocates to the nucleus, where it binds to specific hormone-response elements in DNA.

The actions of hydrocortisone include induction of the synthesis of enzymes involved with gluconeogenesis, mobilization of adipose triacylglycerol, and degradation of muscle
proteins.

Hydrocortisone thus plays a major role in mediating long-term adaptations in the activities of metabolic pathways in response to starvation, sepsis, and stress.

Question 38

DISEASE STATES ASSOCIATED WITH
ABNORMAL ENZYME FUNCTIONING

Answer 38

1. Vitamin Deficiencies
   a. Scurvy
   b. Pellagra
2. Inborn Errors of Metabolism
3. Vitamin-Dependency Diseases
4. alpha1 -Antitrypsin Deficiency
5. Pancreatitis
6. Enzymes as Markers of Disease

Question 39

DISEASE STATES ASSOCIATED WITH
ABNORMAL ENZYME FUNCTIONING
Vitamin Deficiencies

Answer 39

Since vitamins are crucial components of many enzyme cofactors, an inadequate
concentration of one or more of these essential dietary substances can result in
impaired enzymatic activity. Vitamin deficiencies result from inadequate dietary intake or from impaired absorption or recycling of a vitamin. Impaired absorption of vitamins K and B12.

Unlike many of the other vitamin-based cofactors that attach to their respective
enzymes through noncovalent bonds,

Biotin is covalently attached to lysyl residues of the enzymes with which it functions.

Biotin deficiency can result from inadequate activity of the enzyme biotinidase, which normally hydrolyzes the biotinyl-lysyl bond and releases free biotin, thus permitting recycling of biotin when biotin-containing enzymes are degraded.

A deficiency of biotin can also be induced by consumption of raw eggs, which contain avidin, a protein that binds biotin very tightly, thereby preventing absorption of biotin from the gut.

Biotin deficiency reduces the activities
of all four biotin-dependent enzymes: pyruvate carboxylase, acetyl-CoA carboxylase, propionyl-CoA carboxylase, and P-methylcrotonyl-CoA carboxylase.

Dietary deficiencies of particular vitamins usually result from restricted diets. In
each case, there is impaired activity of all of the enzymes that utilize the particular
vitamin-derived cofactor, and ultimately development of a specific vitamin-deficiency disease.

Thus, deficiency of folic acid results in megaloblastic anemia and is associated with congenital neural tube defects, whereas the peripheral neuropathy and cardiac manifestations of beriberi are caused by a dietary deficiency of thiamine.

Question 40

DISEASE STATES ASSOCIATED WITH
ABNORMAL ENZYME FUNCTIONING
Vitamin Deficiencies
Scurvy

Answer 40

Scurvy is the result of a dietary **deficiency of vitamin C **(ascorbic acid), which is usually obtained from fresh fruits and vegetables, especially citrus
fruits (e.g., oranges, grapefruit, limes), cabbage, mangoes, and tomatoes.

Ascorbic acid is a cofactor in various hydroxylation reactions including hydroxylation of specific proline and lysine residues of procollagen and the
hydroxylation of dopamine to form norepinephrine. Scurvy is primarily a disease of defective collagen synthesis, and is characterized by bleeding gums, hemorrhages, and impaired wound heeling.

Ascorbic acid also plays an important non-cofactor role as an antioxidant; it regenerates the reduced forms of other antioxidants, such as vitamin E and glutathione, as well as inactivating potentially harmful reactive oxygen
species and nitrogen radicals.

Scurvy has been recognized since ancient times, and many indigenous cultures are known to have had remedies utilizing local plant sources, including teas brewed from pine needles. Scurvy was particularly rampant among European sailors on long ocean voyages.

Question 41

DISEASE STATES ASSOCIATED WITH
ABNORMAL ENZYME FUNCTIONING
Vitamin Deficiencies
Pellagra

Answer 41

A deficiency of niacin results in pellagra, which is characterized by dermatitis (especially in areas of the skin exposed to sunlight), diarrhea, dementia, and ultimately-if untreated-death.

Since niacin is the vitamin component of NAD+ and NADP+, which are cofactors in numerous oxidation-reduction reac-
tions, it is involved with essentially all of the major metabolic pathways including
glycolysis, P-oxidation of fatty acids, the TCA cycle, electron transport and oxidative phosphorylation, and the synthesis of fatty acids and cholesterol.

Pellagra was endemic in the American Southeast between the two world wars,
primarily among poor mill workers, who consumed a limited diet consisting primarily of corn (maize) and lard.

The niacin in maize has relatively low bioavdability unless the maize is treated with alkali.

Europeans adopted corn as a crop but not the native tradition of grinding corn with lime (calcium oxide or calcium carbonate).

Niacin is unique among the B vitamins in that the dietary requirement for this
vitamin can be partially satisfied by endogenous synthesis of niacin from the essential amino acid tryptophan; unfortunately, maize also happens to be a relatively poor source of tryptophan.

Prevention of pellagra was accomplished through public health measures, particularly the fortification of cereal products (e.g., bread, biscuits, pasta)
with niacin.

Ironically, these measures were delayed for many years because public acceptance of pellagra as a disease of malnutrition was hampered by the eugenics movement, which stereotyped the victims of pellagra in the U.S. South as inherently inferior human beings.

Question 42

DISEASE STATES ASSOCIATED WITH
ABNORMAL ENZYME FUNCTIONING
Inborn Errors of Metabolism

Answer 42

Inborn errors of metabolism are genetic disorders resulting from partial loss of function or from null mutations (complete absence of activity) of genes coding for particular enzymes.

Examples of inborn errors of metabolism include phenylketonuria (PKU, caused by a lack of phenylalanine hydroxylase), medium-chain acyl-CoA
hydrogenase deficiency, and glucose 6-phosphate dehydrogenase deficiency.

Other examples of inborn errors are the lysosomal storage diseases, which result from the loss of function of acid hydrolases required for lysosomal digestion of glycosaminoglycans, glycolipids, sphingomyelin, and glycogen.

Question 43

DISEASE STATES ASSOCIATED WITH
ABNORMAL ENZYME FUNCTIONING
Vitamin-Dependency Diseases

Answer 43

Inborn errors of metabolism often result from the synthesis of mutated enzymes, which have a decreased affinity (increased Km) for their coenzyme or prosthetic group.

In such cases the patient can often be treated successfully with exceptionally
high intakes-or megadoses-of the vitamin precursor.

For example, cystathionine
synthase is a pyridoxal phosphate-dependent enzyme that synthesizes cystathionine from homocysteine and serine. Some forms of cystathionine beta-synthase deficiency are responsive to treatment with pyridoxine (vitamin B6).

Similarly, some persons who are deficient in pyruvate dehydrogenase improve with thiamine therapy.

Question 44

DISEASE STATES ASSOCIATED WITH
ABNORMAL ENZYME FUNCTIONING
alpha1 -Antitrypsin Deficiency

Answer 44

alpha1-Antitrypsin is a plasma glycoprotein that inhibits the activity of elastase and has inhibitory activity against a number of other serine proteases.

Synthesized in and secreted by the liver, alpha1-antitrypsin’s major physiological function is to inhibit elastase released by neutrophils in the lung.

In the absence of this bloodborne protease inhibitor, there is destruction of elastin in pulmonary alveoli, resulting in chronic obstructive pulmonary disease or emphysema.

The disease occurs much earlier and
is more severe in people who smoke, and some people with alpha1-antitrypsin deficiency also develop cirrhosis of the liver.

Question 45

DISEASE STATES ASSOCIATED WITH
ABNORMAL ENZYME FUNCTIONING
Pancreatitis

Answer 45

Pancreatitis is an inflammation of the pancreas which can result from a number
of conditions, including gallstones, chronic alcoholism, and the blockage of the
pancreatic duct that can occur in cystic fibrosis.

Damage to pancreatic cells results in premature activation of digestive proteases within the pancreas and resulting auto-digestion of the pancreas.

Elevated serum levels of pancreatic enzymes, particularly
pancreatic lipase and amylase, are a laboratory-based diagnostic criterion of acute pancreatitis.

Question 46

DISEASE STATES ASSOCIATED WITH
ABNORMAL ENZYME FUNCTIONING
Enzymes as Markers of Disease

Answer 46

Many tissues produce enzymes that are relatively cell-specific.

Because these enzymes are released into the circulation as a result of tissue damage, assays of the levels of certain enzymes in blood can provide useful diagnostic information.

Probably the most widely requested plasma enzyme assays are those for alanine aminotransferase (ALT) and aspartate aminotransferase (AST), both of which are present in high concentration in hepatocytes. When these cells are injured, for example by viral hepatitis or acetaminophen overdose, ALT and AST are released into the blood. As indicated above, elevated serum levels of pancreatic lipase and amylase are indicative of acute pancreatic disease.

The level of the MB isozyme of
creatine kinase (CPK), a marker for myocardial infarction, often rises rapidly in the plasma of a person who has experienced a heart attack. An increased level of the MM- isozyme of CPK is usually indicative of injury to skeletal muscle.

Other examples of tissue-specific enzyme markers of cellular injury include bone-specific alkaline phosphatase (b-ALKP), which serves as a marker of bone turnover in patients with osteoporosis or Paget’s disease, and acid phosphatase, which is elevated in plasma of
patients with metastatic cancer of the prostate.