Biochemistry, 4th Edition P16 pptx

Related Proteins Share a Common Evolutionary Origin Amino acid sequence analysis reveals that proteins with related functions often show a high degree of sequence similarity.. Human myog

substitutions of other amino acids at these positions cannot be tolerated The

number of amino acid differences between two cytochrome c sequences is

propor-tional to the phylogenetic difference between the species from which they are

de-rived Cytochrome c in humans and in chimpanzees is identical; human and

another mammalian (sheep) cytochrome c differ at 10 residues The human

cyto-chrome c sequence has 14 variant residues from a reptile sequence (rattlesnake),

18 from a fish (carp), 29 from a mollusc (snail), 31 from an insect (moth), and

more than 40 from yeast or higher plants (cauliflower)

The Phylogenetic Tree for Cytochrome c Figure 5.20 displays a phylogenetic tree

(a diagram illustrating the evolutionary relationships among a group of organisms)

constructed from the sequences of cytochrome c The tips of the branches are

occu-pied by contemporary species whose sequences have been determined The tree has

been deduced by computer analysis of these sequences to find the minimum

num-ber of mutational changes connecting the branches Other computer methods can

be used to infer potential ancestral sequences represented by nodes, or branch

points, in the tree Such analysis ultimately suggests a primordial cytochrome c

se-quence lying at the base of the tree Evolutionary trees constructed in this manner,

that is, solely on the basis of amino acid differences occurring in the primary

se-quence of one selected protein, show remarkable agreement with phylogenetic

re-lationships derived from more classic approaches and have given rise to the field of

molecular evolution.

Related Proteins Share a Common Evolutionary Origin

Amino acid sequence analysis reveals that proteins with related functions often

show a high degree of sequence similarity Such findings suggest a common

ancestry for these proteins

Oxygen-Binding Heme Proteins Myoglobin and the - and -globin chains of

hemoglobin constitute a set of paralogous proteins Myoglobin, the oxygen-binding

heme protein of muscle, consists of a single polypeptide chain of 153 residues

Hemoglobin,the oxygen transport protein of erythrocytes, is a tetramer composed

of two ␣-chains (141 residues each) and two ␤-chains (146 residues each) These

glo-bin paralogs—myogloglo-bin, -globin, and -globin—share a strong degree of

sequence homology (Figure 5.21) Human myoglobin and the human -globin

chain show 38 amino acid identities, whereas human -globin and human -globin

have 64 residues in common The relatedness suggests an evolutionary sequence of

events in which chance mutations led to amino acid substitutions and divergence in

primary structure The ancestral myoglobin gene diverged first, after duplication of

a primordial globin gene had given rise to its progenitor and an ancestral

hemo-globin gene (Figure 5.22) Subsequently, the ancestral hemohemo-globin gene duplicated

to generate the progenitors of the present-day -globin and -globin genes The

ability to bind O2 via a heme prosthetic group is retained by all three of these

polypeptides

Serine Proteases Whereas the globins provide an example of gene duplication

giving rise to a set of proteins in which the biological function has been highly

con-served, other sets of proteins united by strong sequence homology show more

di-vergent biological functions Trypsin, chymotrypsin (see Chapter 14), and elastase

are members of a class of proteolytic enzymes called serine proteases because of the

central role played by specific serine residues in their catalytic activity Thrombin,

an essential enzyme in blood clotting, is also a serine protease These enzymes show

sufficient sequence homology to conclude that they arose via duplication of a

prog-enitor serine protease gene, even though their substrate preferences are now quite

different

Phe

Cys His

Gly Pro Leu Gly

Arg Gly

Gly Tyr

Trp

Leu Asn Pro Lys Lys Pro Thr Lys Met Phe Gly

Arg

17 18

29 30 32 34

38 41

45 48

59

68 70

76 78 80 82 84

91

10

72 73

Heme

Gly 6

Asn 52

Tyr 74

100

FIGURE 5.19 The sequence of

cyto-chrome c from more than 40 different

species reveals that 28 residues are in-variant These invariant residues are scat-tered irregularly along the polypeptide chain, except for a cluster between

residues 70 and 80 All cytochrome c

polypeptide chains have a cysteine residue at position 17, and all but one have another Cys at position 14 These Cys residues serve to link the heme

prosthetic group of cytochrome c to the

protein.

114 Chapter 5 Proteins: Their Primary Structure and Biological Functions

Neurospora

crassa

Candida

krusei

Debaryomyces

kloeckri

Baker's

yeast

Pig, bovine, sheep

Dog

Bullfrog

Gray whale

Pekin duck Rabbit

Monkey

Gray kangaroo

Pigeon King penguin

Snapping turtle

Pacific lamprey

Chicken, turkey Horse

Puget Sound dogfish Silkworm moth

Screwworm fly

Fruit fly

Human, chimpanzee

Hornworm moth

25

13

7.5 7.5

12

12.5

14.5

12

15

25

3 2

6 5

11

11 6

4

6.5 2.5 3

2 6

2

2.5 3

4 6

6 4 4 2 2 5

7.5

Tuna Bonito Carp

Castor Mungbean

Sunflower

Ala Thr Ala Asn Glu Thr Ala

? Lys Lys Gly Ala Lys Ile Phe Lys Thr ? Cys Ala Gln Cys His Thr Val Glu Gly ? Asp

Val Glu Lys Gly Lys Lys Ile Phe Ile Met Lys Cys Ser Gln Cys His Thr Val Glu Gly Lys Asp

Ala Pro

Ancestral

cytochrome c

Human

cytochrome c

Val Gly Pro Asn Leu His Gly Leu Phe Gly Arg Lys ? Gly Gln Ala ? Gly Tyr Thr Asp Lys

Thr Gly Pro Asn Leu His Gly Leu Phe Gly Arg Lys Thr Gly Gln Ala Pro Gly Tyr Thr Ala Lys

50 40

30

Lys Asn Lys Gly ? ? Trp ? Glu Asn Thr Leu Phe Glu Tyr Leu Glu Asn Pro Tyr Ile Asn

Lys Asn Lys Gly Ile Ile Trp Gly Glu Asp Thr Leu Met Gln Tyr Leu Glu Asn Pro Tyr Pro Asn

Thr Lys Met ? Phe ? Gly Leu Lys Lys ? ? Asp Arg Ala Asp Leu Ile Ala Lys ? Gly

Thr Lys Met Ile Phe Val Gly Lys Lys Lys Glu Glu Arg Ala Asp Leu Ile Ala Lys Lys Gly

Ile

FIGURE 5.20 This phylogenetic tree depicts the evolutionary relationships among organisms as determined

by the similarity of their cytochrome c amino acid sequences The numbers along the branches give the

amino acid changes between a species and a hypothetical progenitor Note that extant species are located

only at the tips of branches Below, the sequence of human cytochrome c is compared with an inferred

an-cestral sequence represented by the base of the tree Uncertainties are denoted by question marks (Adapted

Val His Leu Thr Pro Glu Glu Lys Ser Ala Val Thr Ala Leu Trp Gly Lys Val Asn Val Asp Glu Val Gly Gly Glu Ala

Arg Leu Phe Lys Gly His Pro Glu Thr Leu Glu Lys Phe Asp Lys Phe Lys His Leu Glu Asp Glu Met Lys Ala Ser Glu Ile

Glu

Arg Leu Leu Val Val Tyr Pro Trp Thr Gln Arg Phe Phe Glu Ser Phe Gly Asp Leu Pro Asp Ala Val Met Gly Asn Pro Gly

Lys Lys His Gly Ala Thr Val Leu Thr Ala Leu Gly Gly Ile Leu Lys Lys Lys Gly Glu Ala Glu Ile Lys Pro Leu Ala Leu

Lys Gly His Gly Lys Lys Val Ala Asp Ala Leu Thr Asn Ala Val Ala His Val Asp Pro Asn Ala Leu Ser Ala Leu Ser Val

Lys Ala His Gly Lys Lys Val Leu Gly Ala Phe Ser Asp Gly Leu Ala His Leu Asp Lys Gly Thr Phe Ala Thr Leu Ser Val

His Ala Thr Lys His Lys Ile Pro Val Lys Tyr Leu Glu Phe Ile Ser Glu Cys Ile Val Leu Gln Ser Lys His Pro Gly Ser

His Ala His Lys Leu Arg Val Asp Pro Val Asn Phe Lys Leu Leu Ser His Cys Leu Thr Leu Ala Ala His Leu Pro Ala Leu

His Cys Asp Lys Leu His Val Asp Pro Glu Asn Phe Arg Leu Leu Gly Asn Val Leu Val Leu Ala His His Phe Gly Lys Leu

Gly Ala Asp Ala Gln Gly Ala Met Asn Lys Ala Leu Glu Leu Phe Arg Lys Asp Met Asn Tyr Lys Glu Leu Gly Phe Gln Phe

Thr Pro Ala Val His Ala Ser Leu Asp Lys Phe Leu Ala Ser Val Ser Thr Val Leu Lys Tyr Arg

Phe

Thr Pro Pro Val Gln Ala Ala Tyr Gln Lys Val Val Ala Gly Val Ala Asn Ala Leu Lys Tyr His

Phe

Gly

Hemoglobin



-chain of horse methemoglobin -chain of horse methemoglobin Sperm whale myoglobin

FIGURE 5.21 The amino acid sequences of the globin chains of human hemoglobin and myoglobin show a

strong degree of homology The - and -globin chains share 64 residues of their approximately 140 residues

in common Myoglobin and the -globin chain have 38 amino acid sequence identities.This homology is

fur-ther reflected in these proteins’ tertiary structure.

Myoglobin

Ancestral

-globin Ancestral-globin





Ancestral hemoglobin

Ancestral globin

FIGURE 5.22 This evolutionary tree is inferred from the homology between the amino acid sequences

of the -globin, -globin, and myoglobin chains.

Duplication of an ancestral globin gene allowed the divergence of the myoglobin and ancestral he-moglobin genes Another gene duplication event subsequently gave rise to ancestral  and  forms,

as indicated Gene duplication is an important evo-lutionary force in creating diversity.

116 Chapter 5 Proteins: Their Primary Structure and Biological Functions

Apparently Different Proteins May Share a Common Ancestry

A more remarkable example of evolutionary relatedness is inferred from sequence

homology between hen egg white lysozyme and human milk ␣-lactalbumin,

pro-teins of different biological activity and origin Lysozyme (129 residues) and

-lactalbumin (123 residues) are identical at 48 positions Lysozyme hydrolyzes the

polysaccharide wall of bacterial cells, whereas -lactalbumin regulates milk sugar

(lactose) synthesis in the mammary gland Although both proteins act in reactions involving carbohydrates, their functions show little similarity otherwise Neverthe-less, their tertiary structures are strikingly similar (Figure 5.23) It is conceivable that many proteins are related in this way, but time and the course of evolutionary change erased most evidence of their common ancestry In contrast to this case, the

proteins G-actin and hexokinase (Figure 5.24) share essentially no sequence

homol-C 129 N

Hen egg white lysozyme 123

N

C

Human milk -lactalbumin

FIGURE 5.23 The tertiary structures of hen egg white

lysozyme and human -lactalbumin are very similar.

FIGURE 5.24 The tertiary structures of (a) hexokinase and (b) actin; ADP is bound to both proteins (purple).

ogy, yet they have strikingly similar three-dimensional structures, even though their

biological roles and physical properties are very different Actin forms a filamentous

polymer that is a principal component of the contractile apparatus in muscle;

hex-okinase is a cytosolic enzyme that catalyzes the first reaction in glucose catabolism

A Mutant Protein Is a Protein with a Slightly Different

Amino Acid Sequence

Given a large population of individuals, a considerable number of sequence variants

can be found for a protein These variants are a consequence of mutations in a gene

(base substitutions in DNA) that have arisen naturally within the population Gene

mu-tations lead to mutant forms of the protein in which the amino acid sequence is altered

at one or more positions Many of these mutant forms are “neutral” in that the

func-tional properties of the protein are unaffected by the amino acid substitution Others

may be nonfunctional (if loss of function is not lethal to the individual), and still

oth-ers may display a range of aberrations between these two extremes The severity of the

effects on function depends on the nature of the amino acid substitution and its role

in the protein These conclusions are exemplified by the hundreds of human

hemo-globin variants that have been discovered to date Some of these are listed in Table 5.4

A variety of effects on the hemoglobin molecule are seen in these mutants,

in-cluding alterations in oxygen affinity, heme affinity, stability, solubility, and subunit

interactions between the -globin and -globin polypeptide chains Some variants

show no apparent changes, whereas others, such as HbS, sickle-cell hemoglobin

(see Chapter 15), result in serious illness This diversity of response indicates that

some amino acid changes are relatively unimportant, whereas others drastically

al-ter one or more functions of a protein

5.6 Can Polypeptides Be Synthesized in the Laboratory?

Chemical synthesis of peptides and polypeptides of defined sequence can be

car-ried out in the laboratory Formation of peptide bonds linking amino acids together

is not a chemically complex process, but making a specific peptide can be

chal--chain

-chain

*Hemoglobin variants are often given the geographical name of their origin.

Adapted from Dickerson, R E., and Geis, I., 1983 Hemoglobin: Structure, Function, Evolution and Pathology Menlo Park, CA:

Benjamin/Cummings.

118 Chapter 5 Proteins: Their Primary Structure and Biological Functions

Fmoc

H3C CH3

H3C CH3

DIPCDI (diisopropyl) carbodiimide

Activated amino acid

1 4

8

9

5

2

3

7

6

H

Incoming blocked amino acid

R2

Fmoc blocking

group

R1

H2NCHCNHCHC

R2

Incoming blocked

amino acid

Fmoc

R3 NHCHCOOH

Amino-blocked tripeptidyl-resin particle

Fmoc

R1 NHCHC NHCHCNHCHC

R2

Tripeptidyl-resin particle

R1

H2NCHCNHCHCNHCHC

R2

R3

O

R3

O

Amino-blocked dipeptidyl-resin particle

+

R3 NHCHC O O

Dipeptide-resin particle

N

N

H3C CH3

H3C CH3

N

NH O

C NH

NH

H2N CHC

O

R2

R1

Fmoc

Fmoc removal

R1 NHCHCNHCHC

R2

Aminoacyl-resin particle

O

Diisopropylurea

NHCHC

CH3 C

CH3

CH3

t Butyl group

H2N C R

H

OH

R

H

O C

H3C CH3

H3C CH3

Base

H3C CH3

H3C CH3

DIPCDI

(diisopropyl)

carbodiimide

C N

N

H3C CH3

H3C CH3

DIPCDI

C N

N

Activated amino acid

H3C CH3

H3C CH3

C N

NH

Activated amino acid

H3C CH3

H3C CH3

C N

NH

C NH

NH O

H3C CH3

H3C CH3

Fmoc removal Base

+

1

2

3

4

5

ANIMATED FIGURE 5.25 Solid-phase synthesis of a peptide The

9-fluorenylmethoxycarbonyl (Fmoc) group is an excellent orthogonal blocking group for

the-amino group of amino acids during organic synthesis because it is readily removed

under basic conditions that don’t affect the linkage between the insoluble resin and the

-carboxyl group of the growing peptide chain (inset) N,N-diisopropylcarbodiimide

(DIPCDI) is one agent of choice for activating carboxyl groups to condense with amino

groups to form peptide bonds (1) The carboxyl group of the first amino acid (the

carboxyl-terminal amino acid of the peptide to be synthesized) is chemically attached to

an insoluble resin particle (the aminoacyl-resin particle ) (2) The second amino acid, with

its amino group blocked by a Fmoc group and its carboxyl group activated with DIPCDI,

is reacted with the aminoacyl-resin particle to form a peptide linkage, with elimination

of DIPCDI as diisopropylurea (3) Then, basic treatment (with piperidine) removes the

N-terminal Fmoc blocking group, exposing the N-terminus of the dipeptide for another

cycle of amino acid addition (4) Any reactive side chains on amino acids are blocked by

addition of acid-labile tertiary butyl (tBu) groups as an orthogonal protective functions.

(5) After each step, the peptide product is recovered by collection of the insoluble resin

beads by filtration or centrifugation Following cyclic additions of amino acids, the

com-pleted peptide chain is hydrolyzed from linkage to the insoluble resin by treatment with

HF; HF also removes any tBu protecting groups from side chains on the peptide See this

figure animated at www.cengage.com/login

correct sequences are to be synthesized, the -COOH group of residue x must be

linked to the -NH2group of neighboring residue y in a way that prevents reaction

of the amino group of x with the carboxyl group of y In essence, any functional

groups to be protected from reaction must be blocked while the desired coupling

reactions proceed Also, the blocking groups must be removable later under

condi-tions in which the newly formed peptide bonds are stable An ingenious synthetic

strategy to circumvent these technical problems is orthogonal synthesis An

orthogo-nal system is defined as a set of distinctly different blocking groups—one for

side-chain protection, another for -amino protection, and a third for -carboxyl

pro-tection or anchoring to a solid support (see following discussion) Ideally, any of the

three classes of protecting groups can be removed in any order and in the presence

of the other two, because the reaction chemistries of the three classes are

suffi-ciently different from one another In peptide synthesis, all reactions must proceed

with high yield if peptide recoveries are to be acceptable Peptide formation

be-tween amino and carboxyl groups is not spontaneous under normal conditions (see

Chapter 4), so one or the other of these groups must be activated to facilitate the

reaction Despite these difficulties, biologically active peptides and polypeptides

have been recreated by synthetic organic chemistry Milestones include the

pio-neering synthesis of the nonapeptide posterior pituitary hormones oxytocin and

va-sopressin by Vincent du Vigneaud in 1953 and, in later years, larger proteins such

as insulin (21 A-chain and 30 B-chain residues), ribonuclease A (124 residues), and

HIV protease (99 residues)

Solid-Phase Methods Are Very Useful in Peptide Synthesis

Bruce Merrifield and his collaborators pioneered a clever solution to the problem

of recovering intermediate products in the course of a synthesis The

carboxyl-terminal residues of synthesized peptide chains are covalently anchored to an

in-soluble resin (polystyrene particles) that can be removed from reaction mixtures

simply by filtration After each new residue is added successively at the free

amino-terminus, the elongated product is recovered by filtration and readied for the

next synthetic step Because the growing peptide chain is coupled to an insoluble

resin bead, the method is called solid-phase synthesis The procedure is detailed

in Figure 5.25 This cyclic process is automated and computer controlled so that

the reactions take place in a small cup with reagents being pumped in and

re-moved as programmed

5.7 Do Proteins Have Chemical Groups Other

Than Amino Acids?

Many proteins consist of only amino acids and contain no other chemical groups

The enzyme ribonuclease and the contractile protein actin are two such examples

Such proteins are called simple proteins However, many other proteins contain

var-ious chemical constituents as an integral part of their structure Some of these

con-stituents arise through covalent modification of amino acid side chains in proteins

after the protein has been synthesized Such alterations are called post-translational

modifications. For example, the reaction of two cysteine residues in a protein to

form a disulfide linkage (Figure 4.8b) is a post-translational modification Many of

the prominent post-translational modifications, such as those listed in Table 5.5, can

act as “on–off switches” that regulate the function or cellular location of the

pro-tein Approximately 500 post-translational modifications are listed in the RESID

database accessible through the National Cancer Institute Frederick Advanced

Bio-medical Computing Center at http://www.ncifcrf.gov/RESID.

A common form of post-translational modification not to be found in such a

database or in Table 5.5 is the removal of amino acids from the protein by

prote-olytic cleavage Many proteins localized in specific subcellular compartments have

120 Chapter 5 Proteins: Their Primary Structure and Biological Functions

N-terminal signal sequences that stipulate their proper destination Such signal

se-quences typically are clipped off during their journey Other proteins, such as some hormones or potentially destructive proteases, are synthesized in an inactive form and converted into an active form through proteolytic removal of some of their amino acids

The general term for proteins containing nonprotein constituents is conjugated

proteins(Table 5.6) Because association of the protein with the conjugated group does not occur until the protein has been synthesized, these associations are post-translational as well, although such terminology is usually not applied to these pro-teins (with the possible exception of glycopropro-teins) As Table 5.6 indicates, conju-gated proteins are typically classified according to the chemistry of the nonprotein part If the nonprotein part participates in the protein’s function, it is referred to as

a prosthetic group Conjugation of proteins with these different nonprotein

con-stituents dramatically enhances the repertoire of functionalities available to proteins

5.8 What Are the Many Biological Functions of Proteins?

Proteins are the agents of biological function Virtually every cellular activity is dependent

on one or more particular proteins Thus, a convenient way to classify the enormous number of proteins is to group them according to the biological roles they serve

Figure 5.26 summarizes the classification of proteins found in the human proteome

according to their function

Proteins fill essentially every biological role, with the exception of information storage The ability to bind other molecules (ligands) is common to many proteins Binding proteins typically interact noncovalently with their specific ligands Trans-port proteins are one class of binding proteins TransTrans-port proteins include

mem-Amino Acid Side

Phosphorylation PO3  S, T, Y Hormone receptors,

regulatory enzymes

receptors

ADP-ribosylation ADP-ribose H, R G proteins, eukaryotic

elongation factors

TABLE 5.5 Some Prominent Post-Translational Modifications Found in Proteins

Metalloproteins and Ca2, K, Fe2, Zn2, Covalent to noncovalent Metabolic enzymes, kinases, phosphatases,

Hemoproteins Heme group Covalent or noncovalent Hemoglobin, cytochromes

Flavoproteins FMN, FAD Covalent or noncovalent Electron transfer enzymes

TABLE 5.6 Some Common Conjugated Proteins

Proteome is the complete catalog of proteins

encoded by a genome; in cell-specific terms, a

proteome is the complete set of proteins found

in a particular cell type at a particular time

proteins are a class of binding proteins that uses protein–protein interactions to

re-cruit other proteins into multimeric assemblies whose purpose is to mediate and

co-ordinate the flow of information in cells Catalytic proteins (enzymes) mediate

al-most every metabolic reaction Regulatory proteins that bind to specific nucleotide

sequences within DNA control gene expression Hormones are another kind of

reg-ulatory protein in that they convey information about the environment and deliver

this information to cells when they bind to specific receptors Switch proteins such

as G-proteins can switch between two conformational states—an “on” state and an

“off” state—and act via this conformational switching, as regulatory proteins

Struc-tural proteins give form to cells and subcellular structures The great diversity in

function that characterizes biological systems is based on attributes that proteins

possess

All Proteins Function through Specific Recognition and Binding of Some Target

Molecule Although the classification of proteins according to function has

ad-vantages, many proteins are not assigned readily to one of the traditional groupings

Further, classification can be somewhat arbitrary, because many proteins fit more

than one category However, for all categories, the protein always functions through

Chaperone (159, 0.5%) Cell adhesion (577, 1.9%)

Miscellaneous (1318, 4.3%) Viral protein (100, 0.3%) Transfer/carrier protein (203, 0.7%) Transcription factor (1850, 6.0%)

Nucleic acid enzyme (2308, 7.5%)

Signaling molecule (376, 1.2%)

Receptor (1543, 5.0%)

Kinase (868, 2.8%)

Select regulatory

molecule (988, 3.2%)

Transferase (610, 2.0%)

Synthase and synthetase

(313, 1.0%)

Oxidoreductase (656, 2.1%)

Lyase (117, 0.4%) Ligase (56, 0.2%) Isomerase (163, 0.5%) Hydrolase (1227, 4.0%)

Cytoskeletal structural protein (876, 2.8%) Extracellular matrix (437, 1.4%) Immunoglobulin (264, 0.9%) Ion channel (406, 1.3%) Motor (376, 1.2%) Structural protein of muscle (296, 1.0%) Protooncogene (902, 2.9%) Select calcium-binding protein (34, 0.1%) Intracellular transporter (350, 1.1%) Transporter (533, 1.7%)

Molecular function unknown (12809, 41.7%)

Nucleic acid binding

Enzyme

None

FIGURE 5.26 Proteins of the human genome grouped according to their molecular function The numbers and

percentages within each functional category are enclosed in parentheses Note that the function of more than

40% of the proteins encoded by the human genome remains unknown Considering those of known function,

enzymes (including kinases and nucleic acid enzymes) account for about 20% of the total number of proteins;

nucleic acid–binding proteins of various kinds, about 14%, among which almost half are gene-regulatory proteins

(transcription factors) Transport proteins collectively constitute about 5% of the total; and structural proteins,

another 5%.(Adapted from Figure 15 in Venter, J C., et al., 2001 The sequence of the human genome Science 291:1304–1351.)

122 Chapter 5 Proteins: Their Primary Structure and Biological Functions

specific recognition and binding of some other molecule, although for structural proteins, it is usually self-recognition and assembly into stable multimeric arrays

Protein behavior provides the cardinal example of molecular recognition through

struc-tural complementarity, a fundamental principle of biochemistry that was presented in

Chapter 1

Protein Binding The interaction of a protein with its target usually can be de-scribed in simple quantitative terms Let’s explore the simplest situation in which a

protein has a single binding site for the molecule it binds (its ligand; Chapter 1) If

we treat the interaction between the protein (P) and the ligand (L) as a dissociation reaction:

The equilibrium constant for the reaction as written,

Keq [P][L]/[PL],

is a dissociation constant, because it describes the dissociation of the ligand from

the protein Biochemists typically use dissociation constants (KD) to describe bind-ing phenomena Because brackets ([ ]) denote molar concentrations, dissociation

constants have the units of M.

Typically, the ligand concentration is much greater than the protein concentra-tion Under such conditions, a plot of the moles of ligand bound per mole of pro-tein (defined as [PL]/([P]  [PL])) versus [L] yields a hyperbolic curve known as

a saturation curve or binding isotherm (Figure 5.27).

If we define the fractional saturation of P with L, [PL]/([P]  [PL]), as , a lit-tle algebra yields

  [L]/(KD [L])

Thus, when   0.5,

[L] KD

That is, the concentration of L where half the protein has L bound is equal to the

value of KD The smaller this number is, the better the ligand binds to the protein;

that is, a small KD means that the protein is half-saturated with L at a low

con-centration of L In other words, if KDis small, the protein binds the ligand avidly

Typical KDvalues fall in a range from 103M to 1012M.

The Ligand-Binding Site Ligand binding occurs through noncovalent interac-tions between the protein and ligand The lack of covalent interacinterac-tions means that binding is readily reversible Proteins display specificity in ligand binding because

they possess a specific site, the binding site, within their structure that is

comple-Ligand concentration([L])

1.0

0 0

[L] = KD

 =

0.5

FIGURE 5.27 Saturation curve or binding isotherm.