Based on Profs. Kevin Gardner & Reza Khayat 1

Biochemistry - I

Mondays and Wednesdays 9:30-10:45 AM (MR-1307)

SPRING 2017

Lectures 7-8

Outline and Terms

2

• Enzymes (examples and terminology) • The energy diagram • Rate enhanced by enzymes • Enzyme specificity • Specific/general acid-base catalysis • Michaelis-Menten catalysis • The kinetics of enzymatic catalysis • Kinetic constants • Multisubstrate reactions • Reversible enzyme inhibition • Irreversible enzyme inhibition • Enzyme catalysis and pH • Chymotrypsin • Hexokinase • Feedback inhibition • Allosteric enzymes • Catalysis through enzyme reversible covalent modification • Regulation of glycogen phosphorylase • Protein kinases as regulators

Chapter 6: Enzymes

Enzymes

3

• Catalyze (increase rates of) chemical reactions without being altered at end of reaction

• Can use energy from environment (e.g. ATP hydrolysis)

• A few thousand human genetic diseases are due to specific enzyme defects

• Most known enzymes are proteins

• Ribozymes (RNA molecules that catalyze reactions) may play a more prominent role biology than protein enzymes

• Some enzymes require cofactors (right)

• Cofactors participate in catalysis or stabilize protein structure

• Some enzymes are bound to “coenzyme” (next)

Chapter 6: Enzymes

4

• Coenzymes are organic or metalloorganic molecules that carry functional groups necessary for enzymatic activity

• Some enzymes need cofactors and coenzymes • “Prosthetic group” = coenzyme that is tightly/covalently bound • “Holoenzyme” = polypeptide plus coenzyme/cofactor • “Apoenzyme” = polypeptide only

Chapter 6: Enzymes

Enzymes

Classification of Enzymes

5

• Enzymes are classified by reactions they catalyze

• “ase”suffix

• Convert S (substrate) to P (product), while not being consumed themselves

• Substrate binds to enzyme’s “active site”

Chapter 6: Enzymes

6

Chymotrypsin with substrate (red)

bound to active site

Chapter 6: Enzymes

Proteases

ΔG’0 = change in standard free energy under biological conditions ΔG0 = change in standard free energy under chemical conditions Free energy changes as S → P, ΔG‡ is the activation energy Transition state = “fleeting molecular moment” (e.g. bond breaking) Reaction may go to S or P Two activation energies S→P and S←P

Reaction Energy Diagrams

7

E + S ⇌ ES ⇌ EP ⇌ E + P

P has lower energy ground state

Chapter 6: Enzymes

‡

Effect of Enzymes on Reactions

8

• Enzymes enhance reaction rate by lowering activation energy • “rate limiting step” = highest free energy • Enzymes alter RATES, not equilibria • Complementarity between enzyme and transition state (TS) drives catalysis • Enzyme can form covalent bond with intermediate during catalysis • Enzyme must release product(s) and return to original form

Equilibrium constant (K’eq) ΔG’° = –RT * ln(K’eq)

Arrhenius equation: k = A * exp(–ΔG‡ /RT)

k = rate constant A = number of collisions per second

k = (kT/h) * exp(–ΔG‡ /RT) k = Boltzman constant h = Planck’s constant

ΔG’°

Chapter 6: Enzymes

E + S ⇌ ES ⇌ EP ⇌ E + P

Enzyme rate enhancements (kc/ku)

9

• Rate enhancements 5 to 17 orders of magnitude by enzymes (diffusion becomes rate limiting) • Mechanisms:

• “Binding energy”: non-covalent interactions between E and S (H-bonds, VWD, dispersive, electrostatic…)

• Transient covalent bonds with S, provide alternative lower-energy path • ATP, electrochemical gradient…

Chapter 6: Enzymes

Enzyme Specificity

10

Stickase I impedes reaction

• Imagine that Stickase I and stickase II are imaginary enzymes (E) that break sticks (S) • Enzyme specificity: derived from multiple weak interactions between E and S. • Activation energy is paid for by the binding energy (ΔGM or ΔGB) of the substrate to

the enzyme and the enzyme-substrate complementarity achieved during transition state

Stickase II promotes reaction

Binding energy =ΔGM ΔG‡

cat = Δ‡Guncat - ΔGMChapter 6: Enzymes

Factors Influencing Catalysis

11

•Entropy: •entropic reduction of substrate and enzyme hinder catalysis •entropic increase from desolvation of substrate and enzyme promote catalysis

•Enthalpic: •distortion of substrate hinders catalysis •protein-substrate complementarity promotes catalysis

•Cofactors and prosthetic groups promote catalysis •Metals can mediate oxidation-reduction reactions

•Close vicinity of functional acidic/basic groups from amino acids promote catalysis (increased apparent concentration)

1. concentration is moles/liter 2. higher concentration = more crowded environment 3. functional groups located close to one another in an active site are in a close/crowded

environment 4. thus increased apparent concentration of necessary moieties to conduct catalysis

•Specific vs. general acid/base catalysis

Chapter 6: Enzymes

General Acid/Base Catalysis

12

Definition: 1. Protonation/deprotonation occurs during the rate determining step 2. Requires a weak acid/base

Chapter 6: Enzymes

Theory: Michaelis-Menten Equation

13

• Vmax = maximum velocity

• as ↑[S], changes in rate of S → P decreases, or rate reaches an asymptote because all enzymes are working at full capacity.

Vo =Vmax [S]Km + [S]Vmax = k3[Et] because k3 (kcat) is rate limiting.

KM = (k2 + k3)/k1; Km is dissociation constant when k3 << k2. Else, Km is more complicated...

Chapter 6: Enzymes

overall scheme

E + S ⇌ ES ⟶ E + Pk1

k2

k3

Theory: Michaelis-Menten Equation

14

• Vmax = maximum velocity

• as ↑[S], changes in rate of S → P decreases, or rate reaches an asymptote because all enzymes are working at full capacity.

Vo =Vmax [S]Km + [S]

k3 must be rate limiting for Michaelis-Menten to be a valid model

Vmax = k3[Et] because k3 (kcat) is rate limiting. KM = (k2 + k3)/k1; Km is dissociation constant when k3 << k2. Else, Km is more complicated...

[E]T

We can experimentally vary [ET] and [S]

(d[P]/dt) = k3*[ES]

At saturating [S] all [ET] will be in complex

(d[P]/dt) = k3*[ES]

Km = 1/Ka

=[E]T

Chapter 6: Enzymes

• [ET] is kept constant and [S] is increased

• [S] affects rate of reaction

• [S] is depleted during a reaction

• with ↑ [S] there is an ↑ rate (V)

• Each line is a new experiment

• Vo = initial velocity when [S] >> [E]

• note that Vo is the same at [S] = 100, 150, and 200µM -this is saturating [S]

Kinetics of Enzyme Catalysis

15

• V0 is the initial (1min) linear portion of graph

• Δ[S] is negligible at Vo, so [S] ~ constant

• V decreases with substrate depletion

• velocity units = concentration sec-1

A typical experiment for studying an enzyme that follows Michaelis-Menten kinetics:

Chapter 6: Enzymes

Lineweaver-Burk Plot

16

y = mx + b

Chapter 6: Enzymes

Range of Michaelis constants

17

• Km values are equilibrium constants, akin to dissociation constants • Lower Km = higher affinity for substrate • Values are specific to each enzyme/substrate pair:

Chapter 6: Enzymes

Range of Turnover Numbers

18

• Varies widely according to needs of cell

• Best way to compare enzymes is to compare their specificity constants kcat/KM (catalytic efficiency) purpose of this course

• kcat describes the limiting rate of S → P at saturating [S]

• kcat = “turnover number” = # of molecules of S → P sec-1 at saturating [S]

Chapter 6: Enzymes

Multi-substrate reactions

19

Many enzymes have two or more substrates (Hexokinase + ATP + glucose)

May or may not form “ternary” complex (3 or more partners)

Enzymes200

Many Enzymes Catalyze Reactions with Two or More Substrates

We have seen how [S] affects the rate of a simple enzy-matic reaction (S→ P) with only one substrate mole-cule. In most enzymatic reactions, however, two (andsometimes more) different substrate molecules bind tothe enzyme and participate in the reaction. For exam-ple, in the reaction catalyzed by hexokinase, ATP andglucose are the substrate molecules, and ADP and glu-cose 6-phosphate are the products:

The rates of such bisubstrate reactions can also beanalyzed by the Michaelis-Menten approach. Hexoki-nase has a characteristic Km for each of its substrates(Table 6–6).

Enzymatic reactions with two substrates usually in-volve transfer of an atom or a functional group from onesubstrate to the other. These reactions proceed by oneof several different pathways. In some cases, both sub-strates are bound to the enzyme concurrently at somepoint in the course of the reaction, forming a noncova-lent ternary complex (Fig. 6–13a); the substrates bindin a random sequence or in a specific order. In othercases, the first substrate is converted to product anddissociates before the second substrate binds, so no ter-nary complex is formed. An example of this is the Ping-Pong, or double-displacement, mechanism (Fig. 6–13b).Steady-state kinetics can often help distinguish amongthese possibilities (Fig. 6–14).

ATP ! glucose ¡ ADP ! glucose 6-phosphate

simple rearrangement of Equation 6–26 by dividing bothsides by Vmax gives

Thus, the ratio V0/Vmax " 9.6 !M S#1/12 !M S#1 "[S]/(Km ! [S]). This simplifies the process of solvingfor Km, giving 0.25[S] or 10 !M.

V0

Vmax"

[S]Km ! [S]

WORKED EXAMPLE 6–2 Determining [S]

In a separate happyase experiment using [Et] " 10 nM,the reaction velocity, V0, is measured as 3 !M s#1. Whatis the [S] used in this experiment?

Solution: Using the same logic as in Worked Example 6–1,we see that the Vmax for this enzyme concentration is6 !M s#1. Note that the V0 is exactly half of the Vmax.Recall that Km is by definition equal to the [S] where V0 "1⁄2Vmax. Thus, the [S] in this problem must be the sameas the Km, or 10 !M. If V0 were anything other than1⁄2Vmax, it would be simplest to use the expressionV0/Vmax " [S]/(Km ! [S]) to solve for [S].

Ordered

Random order

E

ES

ES

1

2

S2 P2E ! !

! !

P1

(a)

E ! S1 ES1

S2

P2ES1S2

ES1

E P1

Enzyme reaction involving a ternary complex

(b)

E ! S1 ES1 E P2E !P1

S2

E S2E$$$P1

Enzyme reaction in which no ternary complex is formed

FIGURE 6–13 Common mechanisms for enzyme-catalyzed bisub-strate reactions. (a) The enzyme and both substrates come together toform a ternary complex. In ordered binding, substrate 1 must bindbefore substrate 2 can bind productively. In random binding, the sub-strates can bind in either order. (b) An enzyme-substrate complexforms, a product leaves the complex, the altered enzyme forms a sec-ond complex with another substrate molecule, and the second productleaves, regenerating the enzyme. Substrate 1 may transfer a functionalgroup to the enzyme (to form the covalently modified E$), which issubsequently transferred to substrate 2. This is called a Ping-Pong ordouble-displacement mechanism.

Increasing[S2]

1 V0

1M

/min

(

)%

Increasing[S2]

(b)1

[S1]1

mM( )

(a)1

[S1]1

mM( )

1 V0

1M

/min

(

)%

FIGURE 6–14 Steady-state kinetic analysis of bisubstrate reactions. Inthese double-reciprocal plots (see Box 6-1), the concentration of sub-strate 1 is varied while the concentration of substrate 2 is held con-stant. This is repeated for several values of [S2], generating severalseparate lines. (a) Intersecting lines indicate that a ternary complex isformed in the reaction; (b) parallel lines indicate a Ping-Pong (double-displacement) pathway.

Enzymes200

Many Enzymes Catalyze Reactions with Two or More Substrates

We have seen how [S] affects the rate of a simple enzy-matic reaction (S→ P) with only one substrate mole-cule. In most enzymatic reactions, however, two (andsometimes more) different substrate molecules bind tothe enzyme and participate in the reaction. For exam-ple, in the reaction catalyzed by hexokinase, ATP andglucose are the substrate molecules, and ADP and glu-cose 6-phosphate are the products:

The rates of such bisubstrate reactions can also beanalyzed by the Michaelis-Menten approach. Hexoki-nase has a characteristic Km for each of its substrates(Table 6–6).

Enzymatic reactions with two substrates usually in-volve transfer of an atom or a functional group from onesubstrate to the other. These reactions proceed by oneof several different pathways. In some cases, both sub-strates are bound to the enzyme concurrently at somepoint in the course of the reaction, forming a noncova-lent ternary complex (Fig. 6–13a); the substrates bindin a random sequence or in a specific order. In othercases, the first substrate is converted to product anddissociates before the second substrate binds, so no ter-nary complex is formed. An example of this is the Ping-Pong, or double-displacement, mechanism (Fig. 6–13b).Steady-state kinetics can often help distinguish amongthese possibilities (Fig. 6–14).

ATP ! glucose ¡ ADP ! glucose 6-phosphate

simple rearrangement of Equation 6–26 by dividing bothsides by Vmax gives

Thus, the ratio V0/Vmax " 9.6 !M S#1/12 !M S#1 "[S]/(Km ! [S]). This simplifies the process of solvingfor Km, giving 0.25[S] or 10 !M.

V0

Vmax"

[S]Km ! [S]

WORKED EXAMPLE 6–2 Determining [S]

In a separate happyase experiment using [Et] " 10 nM,the reaction velocity, V0, is measured as 3 !M s#1. Whatis the [S] used in this experiment?

Solution: Using the same logic as in Worked Example 6–1,we see that the Vmax for this enzyme concentration is6 !M s#1. Note that the V0 is exactly half of the Vmax.Recall that Km is by definition equal to the [S] where V0 "1⁄2Vmax. Thus, the [S] in this problem must be the sameas the Km, or 10 !M. If V0 were anything other than1⁄2Vmax, it would be simplest to use the expressionV0/Vmax " [S]/(Km ! [S]) to solve for [S].

Ordered

Random order

E

ES

ES

1

2

S2 P2E ! !

! !

P1

(a)

E ! S1 ES1

S2

P2ES1S2

ES1

E P1

Enzyme reaction involving a ternary complex

(b)

E ! S1 ES1 E P2E !P1

S2

E S2E$$$P1

Enzyme reaction in which no ternary complex is formed

FIGURE 6–13 Common mechanisms for enzyme-catalyzed bisub-strate reactions. (a) The enzyme and both substrates come together toform a ternary complex. In ordered binding, substrate 1 must bindbefore substrate 2 can bind productively. In random binding, the sub-strates can bind in either order. (b) An enzyme-substrate complexforms, a product leaves the complex, the altered enzyme forms a sec-ond complex with another substrate molecule, and the second productleaves, regenerating the enzyme. Substrate 1 may transfer a functionalgroup to the enzyme (to form the covalently modified E$), which issubsequently transferred to substrate 2. This is called a Ping-Pong ordouble-displacement mechanism.

Increasing[S2]

1 V0

1M

/min

(

)%

Increasing[S2]

(b)1

[S1]1

mM( )

(a)1

[S1]1

mM( )

1 V0

1M

/min

(

)%

FIGURE 6–14 Steady-state kinetic analysis of bisubstrate reactions. Inthese double-reciprocal plots (see Box 6-1), the concentration of sub-strate 1 is varied while the concentration of substrate 2 is held con-stant. This is repeated for several values of [S2], generating severalseparate lines. (a) Intersecting lines indicate that a ternary complex isformed in the reaction; (b) parallel lines indicate a Ping-Pong (double-displacement) pathway.

For each series [S2] varied while [S1] constant

Chapter 6: Enzymes

20

• Competitive inhibitors are often structurally similar to substrate • Binds active site and inhibits catalysis • Competes with S for active site • Both S and I “fit” in active site, mixture is composed of ES or EI

KI = [E][I]/[EI]

Vo =Vmax [S]

αKm + [S]

α = 1 + [I]/KI

αKm AKA “apparent Km”

KM = (k2 + k3)/k1

k1

k2

k3

Chapter 6: Enzymes

Competitive Inhibition

time

[pro

duct

]

Example: Competitive inhibitors

21

• Transition state analogs

• Designed to bind enzymes tightly

• May be good inhibitors of enzyme

Chapter 6: Enzymes

22

No effect on Vmax because with ↑ [S], overcomes I Km is modified by factor α Km/Vmax does change

-1/αKm

Chapter 6: Enzymes

Competitive Inhibition

Non-competitive Inhibition

23

• Inhibitor binds to site distinct from active site

• Binds only to ES to form ESI

KI’ = ([ES][I]) / [ESI]

Vo =Vmax [S]Km + α’[S]

α’ = 1 + [I]/KI’

KM = (k2 + k3 + k4)/(k1 + k5)

k1

k2

k3

k4k5

Chapter 6: Enzymes

time

[pro

duct

]

Reversible enyzme inhibition:

24

• I binds to ES and thus lowers both apparent [E] and [S] • Reduction of ES means lower Vmax and lower Km • Km/Vmax (kcat/Km) does not change

-α’/Km

1/Vmax

Chapter 6: Enzymes

X

• I binds site distinct from active site, similar to uncompetitive inhibition • Binds E or ES forming EI or ESI, thus both Vmax and Km are affected • Vmax is conversion of ES to P. Thus it is reduced by α’ • [S] available for catalysis is reduced since some is bound to E. Thus Km is affected by

both α and α’ • α ≠ α’

Mixed-type Inhibition

25

KI’ = ([ES][I]) / [ESI]KI = ([E][I]) /[EI]

Vo =Vmax [S]αKm + α’[S] α’ = 1 + [I]/KI’

α = 1 + [I]/KI

Chapter 6: Enzymes

time

[pro

duct

]

Reversible enyzme inhibition: Mixed-type

26

• Vmax modified by 1/α’ • Km modified by α’/α

-α’/αKm

α’/Vmax

Chapter 6: Enzymes

Chymotrypsin: A Protease

27Chapter 6: Enzymes

• Substrate specificity • Nucleophilic attack of substrate by Ser195 • Tuning of Ser195 reactivity • Acyl enzyme intermediate

Irreversible Enyzme Inhibition

28

• Reaction of chymotrypsin with diisopropylfluorophosphate (DIFP) leads to a covalent modification of the enzyme’s nucleophile (Ser195) to irreversibly inhibit the enzyme

• Ser195 can’t carry out its function, thus enzyme is no longer active

Chapter 6: Enzymes

pH-dependence of Enzyme Catalysis

29

• pH alters the activity of many enzymes • pH alters ionization of R groups, coenzymes and cofactors which in turn may alter the 3D

structure of enzyme or chemistry of active site • Often pH optimum related to biological setting:

• Pepsin is a digestive enzyme in stomach (pH optimum ~ 1.6). pH in gastric juice is ~ 1.5 after eating a meal

• Glucose 6-phosphatase acts in liver cells (pH optimum ~ 7.8), pH in cell ~ 7.2

Chapter 6: Enzymes

Example: pH Dependence of Chymotrypsin

30

•3 sets of disulfide bonds •3 key active site residues (“catalytic triad”) Ser195, His57 and Asp102 are shown as ball and stick

6.4 Examples of Enzymatic Reactions 207

Additional features of the chymotrypsin mechanismhave been discovered by analyzing the dependence of thereaction on pH. The rate of chymotrypsin-catalyzedcleavage generally exhibits a bell-shaped pH-rate profile(Fig. 6–20). The rates plotted in Figure 6–20a are ob-tained at low (subsaturating) substrate concentrationsand therefore represent kcat/Km (see Eqn 6–27, p. 199).The plot can be dissected further by first obtaining themaximum rates at each pH, and then plotting kcat aloneversus pH (Fig. 6–20b); after obtaining the Km at eachpH, researchers can then plot 1/Km (Fig. 6–20c). Kineticand structural analyses have revealed that the change inkcat reflects the ionization state of His57. The decline inkcat at low pH results from protonation of His57 (so that itcannot extract a proton from Ser195 in step 1 of the re-action; see Fig. 6–21). This rate reduction illustrates theimportance of general acid and general base catalysis inthe mechanism for chymotrypsin. The changes in the1/Km term reflect the ionization of the !-amino group ofIle16 (at the amino-terminal end of one of chymotrypsin’sthree polypeptide chains). This group forms a salt bridgeto Asp194, stabilizing the active conformation of the

enzyme. When this group loses its proton at high pH, thesalt bridge is eliminated and a conformational changecloses the hydrophobic pocket where the aromatic aminoacid side chain of the substrate inserts (Fig. 6–18). Sub-strates can no longer bind properly, which is measuredkinetically as an increase in Km.

The nucleophile in the acylation phase is the oxygenof Ser195. (Proteases with a Ser residue that plays this rolein reaction mechanisms are called serine proteases.) ThepKa of a Ser hydroxyl group is generally too high for theunprotonated form to be present in significant concentra-tions at physiological pH. However, in chymotrypsin,Ser195 is linked to His57 and Asp102 in a hydrogen-bondingnetwork referred to as the catalytic triad. When a pep-tide substrate binds to chymotrypsin, a subtle change inconformation compresses the hydrogen bond betweenHis57 and Asp102, resulting in a stronger interaction, calleda low-barrier hydrogen bond. This enhanced interactionincreases the pKa of His57 from !7 (for free histidine) to!12, allowing the His residue to act as an enhanced gen-eral base that can remove the proton from the Ser195 hy-droxyl group. Deprotonation prevents development of avery unstable positive charge on the Ser195 hydroxyl andmakes the Ser side chain a stronger nucleophile. At laterreaction stages, His57 also acts as a proton donor, proto-nating the amino group in the displaced portion of the sub-strate (the leaving group).

As the Ser195 oxygen attacks the carbonyl group of the substrate, a very short-lived tetrahedral interme-diate is formed in which the carbonyl oxygen acquires anegative charge (Fig. 6–21, step 2 ). This charge, form-ing within a pocket on the enzyme called the oxyanionhole, is stabilized by hydrogen bonds contributed bythe amide groups of two peptide bonds in the chy-motrypsin backbone. One of these hydrogen bonds(contributed by Gly193) is present only in this interme-diate and in the transition states for its formation andbreakdown; it reduces the energy required to reachthese states. This is an example of the use of bindingenergy in catalysis.

The role of transition state complementarity inenzyme catalysis is further explored in Box 6–3.

6 7 8pH

9 10

v

kcat

Km

6

1

7 8pH

9 10

6 7 8pH

9 10

(a)

(b)

(c)

FIGURE 6–20 The pH dependence of chymotrypsin-catalyzed reac-tions. (a) The rates of chymotrypsin-mediated cleavage produce a bell-shaped pH-rate profile with an optimum at pH 8.0. The rate (v) beingplotted is that at low substrate concentrations and thus reflects the termkcat/Km. The plot can be broken down to its components by using kineticmethods to determine the terms kcat and Km separately at each pH.When this is done (b and c), it becomes clear that the transition justabove pH 7 is due to changes in kcat, whereas the transition above pH 8.5is due to changes in 1/Km. Kinetic and structural studies have shown thatthe transitions illustrated in (b) and (c) reflect the ionization states of theHis57 side chain (when substrate is not bound) and the !-amino group ofIle16 (at the amino terminus of the B chain), respectively. For optimalactivity, His57 must be unprotonated and Ile16 must be protonated.

• pH activity profile of chymotrypsin

• v measured at appropriate [S] reflects kcat/Km

• kcat can be measured at various pH values

• Graph demonstrates that kcat reaches a plateau at pH 8

• Km can be measured at various pH values

• Graph demonstrates that Km starts at a plateau and decreases at pH 8

Chapter 6: Enzymes

Allosteric Enzymes

31

• Example: threonine dehydratase = 4 identical subunits • Substrate is positive (stimulatory) modulator (recall Hb) • Sigmoidal substrate saturation curve • “Cooperative effects”: binding of S to one subunit alters conformation and enhances

binding of S to subsequent subunits • All four subunits have an active site

Chapter 6: Enzymes

Allosteric enzymes

32

• Allosteric enzyme: control (center)

• “+” = positive allosteric modulator lowers Km

• “-” = negative allosteric modulator increases Km

• No change in Vmax • Sigmoid V vs. S curves

Chapter 6: Enzymes

Control of Enzyme Activity by PTMs

33

1.

2.

• Enzyme activity (On/Off/Variable) is regulated via covalent modification by prosthetic group.

• Modification of enzyme results in structural change(s)

Examples:

• Most common, 1/2 to 1/3 of all eukaryotic proteins, single or multiple sites

• Diphtheria toxin inactivates ribosomal factor 2, inhibits protein synthesis and results in cellular death

Chapter 6: Enzymes

Protein Kinases

34

• Eukaryotic protein kinases phosphorylate specific Ser, Thr, Tyr –OH

• Kinases recognize specific sequences (motifs)

• There may be multiple sites in the same protein, can be randomly or sequentially phosphorylated

• Generally reversible, phosphate added and removed by different enzymes

• Example: at least nine sites in five regions of glycogen synthase (right)

• Modifications are not simple on/off, instead finely tuned modulation (variable) of activity in response to various signals

Chapter 6: Enzymes

Target Sequences of Kinases

35Chapter 6: Enzymes

Selectivity and Diversity

Enzyme

Substrate

Substrate Region

Active Site

Difficult to have a diverse substrate pool

Overlap of Substrate and Recognition Regions

Enzyme

Substrate

Substrate Region

Active Site

Docking Region

Docking Site

Separation of Substrate and Recognition Regions

Easier to have variability without interfering with the substrate region and therefore a more

diverse set of targets with the same/similar substrate region

36

Remote Docking Sites

Activation Loop

N-lobe

C-lobe

Φhyd

Φchg

D-recruitment Site (DRS)D-site: (R/K)2-3-(X)2-6-φA-X-φB

F-recruitment Site (FRS)F-site: F-X-F-P Phosphorylates on a (S/T)P motif 37

STI-571 Background: CML/Bcr-Abl and STI-571 structure

38

STI-571 chemical structure; initial lead identified by screening libraries of known inhibitors to other

kinases and subsequently optimized for specificity and other factors

Bcr-Abl fusion generated by chromosomal rearrangement, leading to protein with constitutive kinase activity and CML

Structural Basis of STI-571 Inhibition of Abl

39Schindler et al., Science 289(2000): 1938

STI-571 locks a critical element of the Abl protein structure (activation loop) into a conformation that is incompatible with catalysis

Effect of Abl Mutations on STI-571 Binding

40

Shah et al., Cancer Cell 2(2002): 117-125

• Note that IC50 is concentration required to get 50% inhibition in a given assay, somewhat different from Ki