Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics 8.5. Enzymes Can Be Inhibited by Specific Molecules

Figure 8.31. Formation of a Penicilloyl-Enzyme Complex. Penicillin reacts with the transpeptidase to form an inactive

complex, which is indefinitely stable.

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics

8.6. Vitamins Are Often Precursors to Coenzymes

Earlier (Section 8.1.1), we considered the fact that many enzymes re- quire cofactors to be catalytically active.

One class of these cofactors, termed coenzymes, consists of small organic molecules, many of which are derived

from vitamins. Vitamins themselves are organic molecules that are needed in small amounts in the diets of some higher

animals. These molecules serve the same roles in nearly all forms of life, but higher animals lost the capacity to

synthesize them in the course of evolution. For instance, whereas E. coli can thrive on glucose and organic salts, human

beings require at least 12 vitamins in the diet. The biosynthetic pathways for vitamins can be complex; thus, it is

biologically more efficient to ingest vitamins than to synthesize the enzymes required to construct them from simple

molecules. This efficiency comes at the cost of dependence on other organisms for chemicals essential for life. Indeed,

vitamin deficiency can generate diseases in all organisms requiring these molecules (Tables 8.9 and 8.10). Vitamins can

be grouped according to whether they are soluble in water or in nonpolar solvents.

8.6.1. Water-Soluble Vitamins Function As Coenzymes

Table 8.9 lists the water-soluble vitamins ascorbic acid (vitamin C) and a series known as the vitamin B

complex (Figure 8.32). Ascorbate, the ionized form of ascorbic acid, serves as a reducing agent (an antioxidant),

as will be discussed shortly. The vitamin B series comprises components of coenzymes. Note that, in all cases except

vitamin C, the vitamin must be modified before it can serve its function.

Vitamin deficiencies are capable of causing a variety of pathological conditions (see Table 8.9). However, many of the

same symptoms can result from conditions other than lack of a vitamin. For this reason and because vitamins are

required in relatively small amounts, pathological conditions resulting from vitamin deficiencies are often difficult to

diagnose.

The requirement for vitamin C proved relatively straightforward to demonstrate. This water-soluble vitamin is not used

as a coenzyme but is still required for the continued activity of proyl hydroxylase. This enzyme synthesizes 4-

hydroxyproline, an amino acid that is required in collagen, the major connective tissue in vertebrates, but is rarely found

anywhere else. How is this unusual amino acid formed and what is its role? The results of radioactive-labeling studies

showed that proline residues on the amino side of glycine residues in nascent collagen chains become hydroxylated. The

oxygen atom that becomes attached to C-4 of proline comes from molecular oxygen, O

. The other oxygen atom of O

taken up by α -ketoglutarate, which is converted into succinate (Figure 8.33). This complex reaction is catalyzed by

prolyl hydroxylase, a dioxygenase. It is assisted by an Fe

ion, which is tightly bound to it and needed to activate O

The enzyme also converts α-ketoglutarate into succinate without hydroxylating proline. In this partial reaction, an

oxidized iron complex is formed, which inactivates the enzyme. How is the active enzyme regenerated? Ascorbate

(vitamin C) comes to the rescue by reducing the ferric ion of the inactivated enzyme. In the recovery process, ascorbate

is oxidized to dehydroascorbic acid (Figure 8.34). Thus, ascorbate serves here as a specific antioxidant.

Primates are unable to synthesize ascorbic acid and hence must acquire it from their diets. The importance of ascorbate

becomes strikingly evident in scurvy. Jacques Cartier in 1536 gave a vivid description of this dietary deficiency disease,

which afflicted his men as they were exploring the Saint Lawrence River:

Some did lose all their strength, and could not stand on their feet. . . . Others also had all their skins spotted with spots of

blood of a purple colour: then did it ascend up to their ankles, knees, thighs, shoulders, arms, and necks. Their mouths

became stinking, their gums so rotten, that all the flesh did fall off, even to the roots of the teeth, which did also almost

all fall out.

James Lind, a Scottish physician, illuminated the means of preventing scurvy in an article titled "A Treatise of the

Scurvy" published in 1747. Lind described a controlled study establishing that scurvy could be prevented by including

citrus fruits in the diet. The Royal Navy eventually began issuing lime rations to sailors, from which custom British

sailors acquired the nickname "limeys." Lind's research was inspired by the plight of an expedition commanded by

Commodore George Anson. Anson left England in 1740 with a fleet of six ships and more than 1000 men and returned

with an enormous amount of treasure, but of his crew only 145 survived to reach home. The remainder had died of

scurvy.

Why does impaired hydroxylation have such devastating consequences? Collagen synthesized in the absence of

ascorbate is less stable than the normal protein. Studies of the thermal stability of synthetic polypeptides have been

especially informative. Hydroxyproline stabilizes the collagen triple helix by forming interstrand hydrogen bonds. The

abnormal fibers formed by insufficiently hydroxylated collagen contribute to the skin lesions and blood-vessel fragility

seen in scurvy.

8.6.2. Fat-Soluble Vitamins Participate in Diverse Processes Such as Blood Clotting and

Vision

Not all vitamins function as coenzymes. The fat-soluble vitamins, which are designated by the letters A, D, E, and

K (Figure 8.35, Table 8.10), have a diverse array of functions. Vitamin K, which is required for normal blood

clotting (K from the German koagulation), participates in the carboxylation of glutamate residues to γ-carboxyglutamate,

which makes modified glutamic acid a much stronger chelator of Ca

(Section 10.5.7). Vitamin A (retinol) is the

precursor of retinal, the light-sensitive group in rhodopsin and other visual pigments (Section 32.3.1). A deficiency of

this vitamin leads to night blindness. In addition, young animals require vitamin A for growth. Retinoic acid, which

contains a terminal carboxylate in place of the alcohol terminus of retinol, serves as a signal molecule and activates the

transcription of specific genes that mediate growth and development (Section 31.3). A metabolite of vitamin D is a

hormone that regulates the metabolism of calcium and phosphorus. A deficiency in vitamin D impairs bone formation in

growing animals. Infertility in rats is a consequence of vitamin E (α-tocopherol) deficiency. This vitamin reacts with and

neutralizes reactive oxygen species such as hydroxyl, radicals before they can oxidize unsaturated membrane lipids,

damaging cell structures.

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics 8.6. Vitamins Are Often Precursors to Coenzymes

Table 8.9. Water-Soluble Vitamins

Vitamin Coenzyme Typical reaction type Consequences of deficiency

Thiamine (B

) Thiamine pyrophosphate Aldehyde transfer Beriberi (weight loss, heart

problems, neurological

dysfunction)

Riboflavin (B

) Flavin adenine dinucleotide

(FAD)

Oxidation-reduction Cheliosis and angular

stomatitus (lesions of the

mouth), dermatitis

Pyridoxine (B

) Pyridoxal phosphate Group transfer to or from

amino acids

Depression, confusion,

convulsions

Nicotinic acid (niacin) Nicotinamide adenine

dinucleotide (NAD

)

Oxidation-reduction Pellagra (dermatitis,

depression, diarrhea)

Pantothenic acid Coenzyme A Acyl-group transfer Hypertension

Biotin Biotin-lysine complexes

(biocytin)

ATP-dependent carboxylation

and carboxyl-group transfer

Rash about the eyebrows,

muscle pain, fatigue (rare)

Folic acid Tetrahydrofolate Transfer of one-carbon

components; thymine

synthesis

Anemia, neural-tube defects in

development

5 -Deoxyadenosyl cobalamin Transfer of methyl groups;

intramolecular rearrangements

Anemia, pernicious anemia,

methylmalonic acidosis

C (ascorbic acid) Antioxidant Scurvy (swollen and bleeding

gums, subdermal hemorrhages)

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics 8.6. Vitamins Are Often Precursors to Coenzymes

Table 8.10. Fat-soluble vitamins

Vitamin Function Deficiency

A Roles in vision, growth, reproduction Night blindness, cornea damage, damage to respiratory and

gastrointestinal tract

D Regulation of calcium and phosphate metabolism Rickets (children): skeletal deformaties, impaired growth

Osteomalacia (adults): soft, bending bones

E Antioxidant Inhibition of sperm production; lesions in muscles and

nerves (rare)

K Blood coagulation Subdermal hemorrhaging

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics 8.6. Vitamins Are Often Precursors to Coenzymes

Figure 8.32. Structures of Some Water-Soluble Vitamins.

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics 8.6. Vitamins Are Often Precursors to Coenzymes

Figure 8.33. Formation of 4-Hydroxyproline. Proline is hydroxylated at C-4 by the action of prolyl hydroxylase, an

enzyme that activates molecular oxygen.

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics 8.6. Vitamins Are Often Precursors to Coenzymes

Figure 8.34. Forms of Ascorbic Acid (Vitamin C). Ascorbate is the ionized form of vitamin C, and dehydroascorbic

acid is the oxidized form of ascorbate.

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics 8.6. Vitamins Are Often Precursors to Coenzymes

Figure 8.35. Structures of Some Fat-Soluble Vitamins.

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics

Summary

Enzymes are Powerful and Highly Specific Catalysts

The catalysts in biological systems are enzymes, and nearly all enzymes are proteins. Enzymes are highly specific and

have great catalytic power. They can enhance reaction rates by factors of 10

or more. Many enzymes require cofactors

for activity. Such cofactors can be metal ions or small, vitamin-derived organic molecules called coenzymes.

Free Energy Is a Useful Thermodynamic Function for Understanding Enzymes

Free energy (G) is the most valuable thermodynamic function for determining whether a reaction can take place and for

understanding the energetics of catalysis. A reaction can occur spontaneously only if the change in free energy (∆ G) is

negative. The free-energy change of a reaction that takes place when reactants and products are at unit activity is called

the standard free-energy change (∆ G°). Biochemists usually use ∆ G°

, the standard free-energy change at pH 7.

Enzymes do not alter reaction equilibria; rather, they increase reaction rates.

Enzymes Accelerate Reactions by Facilitating the Formation of the Transition State

Enzymes serve as catalysts by decreasing the free energy of activation of chemical reactions. Enzymes accelerate

reactions by providing a reaction pathway in which the transition state (the highest-energy species) has a lower free

energy and hence is more rapidly formed than in the uncatalyzed reaction.

The first step in catalysis is the formation of an enzyme-substrate complex. Substrates are bound to enzymes at active-

site clefts from which water is largely excluded when the substrate is bound. The specificity of enzyme-substrate

interactions arises mainly from hydrogen bonding, which is directional, and the shape of the active site, which rejects

molecules that do not have a sufficiently complementary shape. The recognition of substrates by enzymes is

accompanied by conformational changes at active sites, and such changes facilitate the formation of the transition state.

The Michaelis-Menten Model Accounts for the Kinetic Properties of Many Enzymes

The Michaelis-Menten model accounts for the kinetic properties of some enzymes. In this model, an enzyme (E)

combines with a substrate (S) to form an enzyme-substrate (ES) complex, which can proceed to form a product (P) or to

dissociate into E and S.

The rate V

of formation of product is given by the Michaelis-Menten equation:

in which V

max

is the reaction rate when the enzyme is fully saturated with substrate and K

, the Michaelis constant, is

the substrate concentration at which the reaction rate is half maximal. The maximal rate, V

max

, is equal to the product of

or k

cat

and the total concentration of enzyme. The kinetic constant k

cat

, called the turnover number, is the number of

substrate molecules converted into product per unit time at a single catalytic site when the enzyme is fully saturated with

substrate. Turnover numbers for most enzymes are between 1 and 10

per second. The ratio of k

cat

provides a

penetrating probe into enzyme efficiency.

Allosteric enzymes constitute an important class of enzymes whose catalytic activity can be regulated. These enzymes,

which do not conform to Michaelis-Menton kinetics, have multiple active sites. These active sites display cooperativity,

as evidenced by a sigmoidal depen-dence of reaction velocity on substrate concentration.

Enzymes Can Be Inhibited by Specific Molecules

Specific small molecules or ions can inhibit even nonallosteric enzymes. In irreversible inhibition, the inhibitor is

covalently linked to the enzyme or bound so tightly that its dissociation from the enzyme is very slow. Covalent

inhibitors provide a means of mapping the enzyme's active site. In contrast, reversible inhibition is characterized by a

rapid equilibrium between enzyme and inhibitor. A competitive inhibitor prevents the substrate from binding to the

active site. It reduces the reaction velocity by diminishing the proportion of enzyme molecules that are bound to

substrate. In noncompetitive inhibition, the inhibitor decreases the turnover number. Competitive inhibition can be

distinguished from noncompetitive inhibition by determining whether the inhibition can be overcome by raising the

substrate concentration.

The essence of catalysis is selective stabilization of the transition state. Hence, an enzyme binds the transition state more

tightly than the substrate. Transition-state analogs are stable compounds that mimic key features of this highest-energy

species. They are potent and specific inhibitors of enzymes. Proof that transition-state stabilization is a key aspect of

enzyme activity comes from the generation of catalytic antibodies. Transition-state analogs are used as antigens, or

immunogens, in generating catalytic antibodies.

Vitamins Are Often Precursors to Coenzymes

Vitamins are small biomolecules that are needed in small amounts in the diets of higher animals. The water-soluble

vitamins are vitamin C (ascorbate, an antioxidant) and the vitamin B complex (components of coenzymes). Ascorbate is

required for the hydroxylation of proline residues in collagen, a key protein of connective tissue. The fat-soluble

vitamins are vitamin A (a precursor of retinal), D (a regulator of calcium and phosphorus metabolism), E (an antioxidant

in membranes), and K (a participant in the carboxylation of glutamate).

Key Terms

enzyme

substrate

cofactor

apoenzyme

holoenzyme

coenzyme

prosthetic group

free energy

transition state

free energy of activation

active site

induced fit

(the Michaelis constant)

max

Michaelis-Menten equation

turnover number

cat

sequential displacement reaction

double-displacement (Ping-Pong) reaction

allosteric enzyme

competitive inhibition

noncompetitive inhibition

group-specific reagent

affinity label

mechanism-based (suicide) inhibition

transition-state analog

catalytic antibody (abzyme)

vitamin

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics

Appendix: V

max

and K

Can Be Determined by Double-Reciprocal Plots

Before the availability of computers, the determination of K

and V

max

values required algebraic manipulation of the

basic Michaelis-Menten equation. Because V

max

is approached asymptotically (see Figure 8.11), it is impossible to

obtain a definitive value from a typical Michaelis-Menten plot. Because K

is the concentration of substrate at V

max

/2,

it is likewise impossible to determine an accurate value of K

. However, V

max

can be accurately determined if the

Michaelis-Menten equation is transformed into one that gives a straight-line plot. Taking the reciprocal of both sides of

equation 23 gives

A plot of 1/V

versus 1/[S], called a Lineweaver-Burk or double-reciprocal plot, yields a straight line with an intercept

of 1/V

max

and a slope of K

max

(Figure 8.36). The intercept on the x-axis is -1/K

Double-reciprocal plots are especially useful for distinguishing between competitive and noncompetitive inhibitors. In

competitive inhibition, the intercept on the y-axis of the plot of 1/V

versus 1/[S] is the same in the presence and in the

absence of inhibitor, although the slope is increased (Figure 8.37). That the intercept is unchanged is because a

competitive inhibitor does not alter V

max

. At a sufficiently high concentration, virtually all the active sites are filled by

substrate, and the enzyme is fully operative. The increase in the slope of the 1/V

versus 1/[S] plot indicates the strength

of binding of competitive inhibitor. In the presence of a competitive inhibitor, equation 31 is replaced by

in which [I] is the concentration of inhibitor and K

is the dissociation constant of the enzyme-inhibitor complex.

In other words, the slope of the plot is increased by the factor (1 + [I]/K

) in the presence of a competitive inhibitor.

Consider an enzyme with a K

of 10

-4

M. In the absence of inhibitor, V

= V

max

/2 when [S] = 10

-4

M. In the presence

of 2 × 10

-3

M competitive inhibitor that is bound to the enzyme with a K

of 10

-3

M, the apparent K

app

) will be

equal to K

× (1 + [I]/K

), or 3 × 10

-4

M. Substitution of these values into equation 23 gives V

= V

max

/4, when [S] =

-4

M. The presence of the competitive inhibitor thus cuts the reaction rate in half at this substrate concentration.

In noncompetitive inhibition (Figure 8.38), the inhibitor can combine with either the enzyme or the enzyme-substrate

complex. In pure noncompetitive inhibition, the values of the dissociation constants of the inhibitor and enzyme and of

the inhibitor and enzyme-substrate complex are equal (Section 8.5.1). The value of V

max

is decreased to a new value

called V

app

max

, and so the intercept on the vertical axis is increased. The new slope, which is equal to K

app

max

, is

larger by the same factor. In contrast with V

max

, K

is not affected by pure noncompetitive inhibition. The maximal

velocity in the presence of a pure noncompetitive inhibitor, V

max

, is given by

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics Appendix: V

max

and K

Can Be Determined by Double-Reciprocal Plots

Figure 8.36. A Double-Reciprocal or Lineweaver-Burk Plot. A double-reciprocal plot of enzyme kinetics is generated

by plotting 1/V

as a function 1/[S]. The slope is the K

max

, the intercept on the vertical axis is 1/V

max

, and the

intercept on the horizontal axis is -1/K

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics Appendix: V

max

and K

Can Be Determined by Double-Reciprocal Plots

Figure 8.37. Competitive Inhibition Illustrated on a Double-Reciprocal Plot. A double-reciprocal plot of enzyme

kinetics in the presence (

) and absence ( ) of a competitive inhibitor illustrates that the inhibitor has no effect

on V

max

but increases K

I. The Molecular Design of Life 8. Enzymes: Basic Concepts and Kinetics Appendix: V

max

and K

Can Be Determined by Double-Reciprocal Plots

Figure 8.38. Noncompetitive Inhibition Illustrated on a Double-Reciprocal Plot. A double-reciprocal plot of enzyme

kinetics in the presence (

) and absence ( ) of a noncompetitive inhibitor shows that K

is unaltered and V

max

is decreased.