3-D Structure / Function Relationship

Myoglobin/Hemoglobin

First protein structures determined Oxygen carriers Hemoglobin transport O2 from lungs to tissues

Myoglobin O2 storage protein

Mb and Hb subunits structurally similar

8 alpha-helices Contain heme group Mb monomeric protein Hb heterotetramer (a2b2) myoglobin

hemoglobin

Heme group
Functional Heme gp = Fe++ bound to tetrapyrrole ring (protoporphyrin IX complex)
Heme non-covalently bound to globin proteins through His residue

O2 binds non-covalently to heme Fe++, stabilized through H-bonding with another His residue
Heme group located in hydrophobic crevice of globin protein

Oxygen Binding Curves

Mb has hyperbolic O2 binding curve Mb binds O2 tightly. Releases at very low pO2

Hb has sigmoidal O2 binding curve

Hb high affinity for O2 at high pO2 (lungs)

Hb low affinity for O2 at low pO2 (tissues)

O2 Binding to Hb shows positive cooperativity

Hb binds four O2 molecules O2 affinity increases as each O2 molecule binds Increased affinity due to conformation change Deoxygenated form = T (tense) form = low affinity Oxygenated form = R (relaxed) form = high affinity

O2 Binding to Hb shows positive cooperativity

T-conformation

R-conformation

O2 Binding induces conformation change

Heme moves 0.34 nm

Exposing crystal of deoxy-form to air cause crystal to crack

Allosteric Interactions
Allosteric interaction occur when specific molecules bind a protein and modulate activity Allosteric modulators or allosteric effectors Bind reversibly to site separate from functional binding or active site Modulation of activity occurs through change in protein conformation 2,3 bisphosphoglycerate (BPG), CO2 and protons are allosteric effectors of Hb binding of O2

Bohr Effect: Relationship of Hb O2 affinity to PCO2

levels and pH Increased CO2 leads to decreased pH CO2 + H2O <-> HCO3- + H+ At decreased pH several key AAs protonated, causes Hb to take on T-conformation (low affinty) In R-form same AAs deprotonated, form charge charge interactions with positive groups, stabilize Rconformation (High affinity) HCO3- combines with N-terminal alpha-amino group to form carbamate group. --N3H+ + HCO3- --NHCOO Carbamation stabilizes Tconformation

 BPG involved acclimation to high altitude Binding of BPG to Hb causes low O2 affinity BPG binds in the cavity between beta-Hb subunits

Bisphosphoglycerate (BPG)

Stabilizes T-conformation Fetal Hb (a2g2) has low affinity for BPG, allows fetus to compete for O2 with mothers Hb (a2b2) in placenta.

Mutations in a- or b-globin genes can cause disease state

Sickle cell anemia Glu6 to Val6 in b-chain

Causes V6 to bind to hydrophobic pocket in deoxyHb

Polymerizes to form long filaments Cause sickling of cells Sickle cell trait offers advantage against malaria Fragile sickle cells can not support parasite

r a d d i t i o n a l d e t a i l s s e e : D y k e s , G . , C r e p e a u , R . H . a n d E d e l s t e i n , S . J . ( 1 9 7 8 ) . N a t u

Enzymes: Introduction

Catalyst
substance that increase rates of a chemical reaction does not effect equilibrium remain unchanged in overall process reactants bind to catalyst, products are released

Catalysts increase product formation by (1) lowering the energy barrier (activation energy) for the product to form (2) increases the favorable orientation of colliding reactant molecules for product formation to be successful (stabilize transition state intermediate)

Catalytic Power
Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates! Urease is a good example:
Catalyzed rate: 3x104/sec Uncatalyzed rate: 3x10 -10/sec Ratio is 1x1014 !

Specificity
Enzymes selectively recognize proper substrates over other molecules Enzymes produce products in very high yields - often much greater than 95% Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield

Classes of enzymes
1. Oxidoreductases = catalyze oxidationreduction reactions (NADH) 2. Transferases = catalyze transfer of functional groups from one molecule to another. 3. Hydrolases = catalyze hydrolytic cleavage 4. Lyases = catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement. 5. Isomerases = catalyze intramolecular rearrangement. 6. Ligases = catalyze reactions in which two molecules are joined. Enzymes named for the substrates and type of reaction

Co-enzymes
Non-protein molecules that help enzymes function Associate with active site of enzyme Enzyme + Co-enzyme = holoenzyme Enzyme alone = apoenzyme Organic co-enzymes thiamin, riboflavin, niacin, biotin Inorganic co-enzymes Mg ++, Fe++, Zn++, Mn++

Kinetics
study of reaction rate determines number of steps involved determines mechanism of reaction identifies rate-limiting step

 Enzymes: Special proteins that act as biological catalysts

1) Globular proteins with specialized 3-D shapes 2) Lower activation energy by: a) bringing two substrates together (greater chance to react) b) stressing bonds of a substrate (to break them) Enzymes are not changed or consumed by the reaction!

Substrate: The reactant/s that the catalyst or enzyme binds with to in turn speed up the reaction.
Catalyst: substance that stresses chemical bonds & speeds up reaction in forward and/or reverse directions. Catalysts lower the activation energy needed for the reaction to occur.

Activation Energy: energy to start a reaction. Requires energy input to break or form bonds

Mechanism of Enzyme Catalysis

Substrate specific: each enzyme catalyzes only one reaction (works on only 1-2 substrate/s) Active site: surface shape or cleft on the enzyme to which the substrate must fit like a lock and key (Lock and Key theory).

Induced fit: enzyme changes shape of active site so substrate fits perfectly (conformation of substrate and enzyme is only complementary after binding).

1. Proteases: Protein-digesting enzymes added to detergent to remove protein stains from clothes. 2. Pectinases: Enzyme that removes pectin from crushed fruit. Keeps juice from gelling, keeps it liquid for easier removal. Helps remove solids from juice.

Factors affecting enzyme activity

a) Temperature: Disrupts hydrogen bonds, alters protein shape (denature). The enzyme becomes denatured; the shape of its active site is altered and substrate cannot fit. The rate of reaction decreases

b) pH: hydrogen ion concentration disrupts bonds between amino acids

c) Substrate Concentration: Increased substrate concentration increases reaction rate until all enzymes are involved, then reactions level out d) Enzyme Concentration: Increased enzyme concentration increases reaction rate until all substrate is used up, then reactions decrease.

Enzyme Kinetics

Rate constants and reaction order

Rate constant (k) measures how rapidly a rxn occurs A k1 k-1 B + C

Rate (v, velocity) = (rate constant) (concentration of reactants) v= k1 [A] 1st order rxn (rate dependent on concentration of 1 reactant) v= k-1[B][C] 2nd order rxn (rate dependent on concentration of 2 reactants) Zero order rxn (rate is independent of reactant concentration)

+ S

E S

E+S

k1 k-1

ES

k2 k-2

E+P

Initial Velocities
Hold [E] constant [E]<<<<<[S]

[P]
D[P]/DT = Vo1 mM

[S] = 1 mM

time

Initial Velocities
D[P]/DT = Vo10 mM

[S] = 10 mM

[P]

D[P]/DT = Vo5 mM D[P]/DT = Vo1 mM

[S] = 5 mM

[S] = 1 mM

time

Plot Vo vs. [S]

Vo 10 mM
Vo 5 mM

Vo 1 mM

 Michaelis-Menten Equation Describes rectangular hyperbolic plot Vo = Vmax [S] Km + [S]

Vmax = velocity where all of the enzyme is bound to substrate (enzyme is saturated with S)

Km = [S] @ Vmax (units moles/L=M) (1/2 of enzyme bound to S)

Michael-Menten Equation based on three key assumptions

1)

[S] is very large compared to [E], such that when all E is bound in the form of ES, there is still an excess of the S

2)

Initial Velocity Assumption

Measurements made to measure initial velocity (vo). At vo very little product formed. Therefore, the rate at which E + P react to form ES is negligible and k-2 is 0. Therefore

+ S

E S

E+S

k1 k-1

ES

k2 k-2

E+P

Initial Velocities
D[P]/DT = Vo10 mM

[S] = 10 mM

[P]

D[P]/DT = Vo5 mM D[P]/DT = Vo1 mM

[S] = 5 mM

[S] = 1 mM

time

3)

Steady State Assumption

Steady state Assumption = [ES] is constant. The rate of ES formation equals the rate of ES breakdown

+ S

E S

E+S

k1 k-1

ES

k2

E+P

Rate of ES formation
E

+ S

E S

E+S

k1

ES

Rate = k1 [E] [S]

Rate of ES breakdown
E S
ES
k2

E
E+P

E S
ES k-1

E
E+S

+ S

Rate = (k2 [ES]) + (k-1[ES]) Rate = [ES](k2 + k-1)

Thereforeif the rate of ES formation equals the rate of ES breakdown 1) k1[E][S] = [ES](k-1+ k2)

2) (k-1+ k2) / k1 = [E][S] / [ES]

3) (k-1+ k2) / k1 = Km (Michaelis constant)

What does Km mean?

1. Km = [S] at Vmax 2. Km is a combination of rate constants describing the formation and breakdown of the ES complex 3. Km is usually a little higher than the physiological [S]

4. Km represents the amount of substrate required to bind of the available enzyme (binding constant of the enzyme for substrate) 5. Km provides a measure of the affinity of an enzyme for its substrate 6. Km can be used to evaluate the specificity of an enzyme for a substrate (if obeys M-M) 7. Small Km means tight binding (high affinity); high Km means weak binding (low affinity)
Hexose Kinase Glucose + ATP <-> Glucose-6-P + ADP

Glucose Allose Mannose

Km = 8 X 10-6 Km = 8 X 10-3 Km = 5 X 10-6

What does kcat mean?

1. kcat is the 1st order rate constant describing ES E+P 2. Also known as the turnover # because it describes the number of rxns a molecule of enzyme can catalyze per second under optimal condition. 3. Most enzyme have kcat values between 102 and 103 s-1 4. For simple reactions k2 = kcat , for multistep rxns kcat = rate limiting step E+S k1 k-1 ES kcat E+P

What does kcat/Km mean?

It measures how the enzyme performs when S is low

kcat/Km describes an enzymes preference for different substrates = specificity constant

The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together (108 to 109 m-1 s-1)

Catalytic perfection when kcat/Km = diffusion rate

More physiological than kcat

Limitations of M-M
1. Some enzyme catalyzed rxns show more complex behavior E + S<->ES<->EZ<->EP<-> E + P With M-M can look only at rate limiting step Often more than one substrate E+S1<->ES1+S2<->ES1S2<->EP1P2<-> EP2+P1<-> E+P2 Must optimize one substrate then calculate kinetic parameters for the other Assumes k-2 = 0 Assume steady state conditions

2.

3. 4.

Michaelis-Menten
E+S k1 k-1 ES

kcat

E+P

Vo = Vmax [S] Km + [S] Vmax Km Kcat Kcat/Km

Vmax Km kcat kcat/Km

How do you get values for Vmax, Km and kcat?

Can determine Km and Vmax experimentally Km can be determined without an absolutely pure enzyme Kcat values can be determined if Vmax is known and the absolute concentration of enzyme is known (Vmax = kcat[Etotal]

Lineweaver-Burk Plots
(double reciprocal plots)
Plot 1/[S] vs 1/Vo L-B equation for straight line X-intercept = -1/Km Y-intercept = 1/Vmax Easier to extrapolate values w/ straight line vs hyperbolic curve

V max
0.25

B
0.2

B B B

0.15
Vo

0.1

B B

[S] 0.5 0.75 2 4 6 8 10

Vo 0.075 0.09 0.152 0.196 0.21 0.214 0.23

0.05

Km

Km ~ 1.3 mM
5 6 [S] 7 8 9 10

0 0 1 2 3 4

Vmax ~ 0.25

14 12

B B
[S] 2.000 1.333 0.500 0.250 0.167 0.125 0.100 Vo 13.333 11.111 6.579 5.102 4.762 4.673 4.348

10 8

Vo

6 4 2 0 -1

B B B B B

B
-0.5 0 0.5 [S] 1 1.5 2

-1/Km = -0.8 Km = 1.23 mM 1/Vmax = 4.0 Vmax = 0.25

Enzyme Inhibition
Inhibitor substance that binds to an enzyme and interferes with its activity Can prevent formation of ES complex or prevent ES breakdown to E + P. Irreversible and Reversible Inhibitors

Irreversible inhibitor binds to enzyme through covalent bonds (binds irreversibly)

Reversible Inhibitors bind through non-covalent interactions (disassociates from enzyme) Why important?

Reversible Inhibitors
E + S <-> ES -> E + P E + I <-> EI Ki = [E][I]/[EI] Competitive Uncompetitive Non-competitive

Types of Reversible Enzyme Inhibitors

Competitive Inhibitor (CI)

CI binds free enzyme Competes with substrate for enzyme binding. Raises Km without effecting Vmax Can relieve inhibition with more S

Competitive Inhibitors look like substrate

O HO C NH2
O H2N S O NH2

PABA

Sulfanilamide

PABA precursor to folic acid in bacteria O2C-CH2-CH2-CO2 -------> O2C-CH=CH-CO2

succinate fumarate

Succinate dehydrogenase

O2C-CH2-CO2 Malonate

Uncompetitive Inhibitor (UI)

UI binds ES complex Prevents ES from proceeding to E + P or back to E + S. Lowers Km & Vmax, but ratio of Km/Vmax remains the same Occurs with multisubstrate enzymes

Non-competitive Inhibitor (NI)

NI can bind free E or ES complex Lowers Vmax, but Km remains the same NIs dont bind to S binding site therefore dont effect Km Alters conformation of enzyme to effect catalysis but not affect substrate binding

Irreversible Inhibitors
CH3 H C CH3 O F P O O CH3 C CH3 H
H3C O S P O CH3 S O C C CH2 C O O CH2CH3 O H CH2CH3

Diisopropyl fluorophosphate (nerve gas)

malathion

S H3C O P O CH3 S NO2

parathion

Organophosphates Inhibit serine hydrolases Acetylcholinesterase inhibitors

Kinetics of Multisubstrate Reactions

E + A + B <-> E + P + Q

Sequential Reactions a) ordered

b) random
Ping-pong Reactions Cleland Notation

Sequential Reactions
Ordered A E A Random EA E EB (EAB)(EPQ) EP EA B B (EAB) (EPQ) P P Q EQ E Q

EQ
E

Ping-Pong Reactions
A
E

P
(F)

Q
E

(EA)(FP)

(FB)(EQ)

In Ping-Pong rxns first product released before second substrate binds

When E binds A, E changes to F

When F binds B, F changes back to E

Lineweaver-Burk Plot of Multisubstrate Reactions

Sequential
Increasing [B] Ping-Pong

Increasing [B]

1/Vo

1/Vo

1/[S]
Vmax doesnt change Km changes

1/[S] Both Vmax & Km change

Enzyme inhibition
1. 2. 3. Enzyme inhibitors change shape of active site and shut off activity Different types of reversible enzyme inhibition classified based on analysis of Lineweaver-Bulk plots Competitive inhibitors: Competitor molecule binds at active site, prevents substrate from binding. Uncompetitive inhibitor: Inhibitor binds only to ES complex at a site distinct from the active site (allosteric site) Non-competitive inhibitors: Inhibitor binds both to the free enzyme and to the ES complex at the allosteric site which is distinct from the active site.(different site), changes shape of active site. Substrate can't bind.

Irreversible competitive inhibitors

These bind covalently or so tightly to the active site that the enzyme is inactivated irreversibly. Irreversible inhibition doe not obey MichaelisMenten Kinetics
1. Affinity label: Are substrate analogs that posses a highly reactive group that is not present on the natural substrate. Action: It permanently blocks the active site from substrate binding because the group reacts covalently with an amino acid residue. The residue that is modified is not necessarily involved in catalysis. 2. Mechanism-based/suicide inhibitors: Are substrate analogs that are transformed by the catalytic action of the enzyme Action: Their structures are such that the product of this reaction is highly reactive and subsequently combines with an amino acid

residue in the active site, thus inactivating the enzyme 3. Transition-state analogs: Are substrate analogs whose structures closely resemble the transition state of the natural substrate. Action: They do not covalently modify the enzyme but bind the active site so tightly that they irreversibly inactivate it. Medical relevance of enzyme inhibitors 1. Toxicity: Many highly toxic naturally occurring and man-made compounds are irreversible inhibitors e.g nerve gas, sarin, is an acetylcholine esterase inhibitor 2. Therapeutic applications: The rational design of therapeutic drugs often involves the synthesis of inhibitors of certain enzymes e.g fluorouracil. Natural compound used as drugs can also inhibit enzymes (e.g penicillin)

Regulation of Enzymes
1. A change in pH can alter the rates of enzyme-catalysed reactions. Many enzyme exhibit a bell-shapes curve when enzyme activity is plotted against pH. Changes in pH influence: a) The ionization state of the substrate or the enzyme-binding site for substrate b) The ionization state of the catalytic site (active site) of the enzyme c) Protein molecule so that their conformation and catalytic activity change

2.

Temperature: The rate of an enzyme-catalyzed reaction often increases with increasing temperature up to optimum point, then it decreases because enzymes are thermo-labile Product inhibition: Sometimes when the product accumulates, it can inhibit some enzymes. This type of control limits the rate of formation of the product when the product is underused. Covalent modification (several types)
a) Phosphorylation (Effect on enzyme activity): In some enzymes, addition of a phosphate group to a specific amino acid residue (serine, tyrosine or threonine) by specific protein kinases dramatically enhances or depresses activity.
NB: Modification is reversible; phosphorylated enzyme can be dephosphorylated by specific phosphatases.

3.

4.

b)

Nucleotidylation (effect on enzyme): The activities of certain enzymes are regulated by the reversible addition of a nucleotide (e.g adenosine) to a specific amino acid. -Modification is reversible e.g an adenylated enzyme can be deadenylated by a specific enzyme

c)

Proteolytic cleavage: Certain enzymes are synthesised as proenzymes, or zymogens, which are inactive forms of enzymes that become active after being cleaved at a specific site in their polypeptide chain by specific proteases. Examples: Digestive enzymes that hydrolyze proteins (trypsin and pepsin) are synthesized as zymogens in the stomach and pancreas Blood clotting is mediated by a series of proteolytic zymogen activities of several serum enzymes

d)

Allosteric regulation of metabolic pathways: The activity of enzymes that catalyse key regulatory reactions of metabolic pathways are often subject to allosteric regulation. Their activity can be modulated by the binding of allosteric effectors to a site on the enzyme that is distinct from the active sites (i.e, allosteric site). Effectors are positive if they enhance the rate of a reaction (i.e, activators) and negative if they decrease the rate of reaction (i.e inhibitors) Feedback inhibition is negative modulation of the committed step of a metabolic pathway by its end product. This prevents unnecessary production of an excess of end product by shutting down the pathway until more is needed

Enzyme Regulation

Regulation of Enzyme Activity

Enzyme quantity regulation of gene expression (Response time = minutes to hours)

a)
b) c)

Transcription
Translation Enzyme turnover

Enzyme activity (rapid response time = fraction of seconds) a) b) Allosteric regulation Covalent modification

c)
d)

Association-disassociation
Proteolytic cleavage of proenzyme

Allosteric Regulation
End products are often inhibitors Allosteric modulators bind to site other than the active site

Allosteric enzymes usually have 4o structure

Vo vs [S] plots give sigmoidal curve for at least one substrate Can remove allosteric site without effecting enzymatic action

Regulation of Enzyme Activity (biochemical regulation)

1st committed step of a biosynthetic pathway or enzymes at pathway branch points often regulated by feedback inhibition. H 4 I 3 5 J

1 A 2 C B X

3X E 4 F

Efficient use of biosynthetic precursors and energy

Phosphofructokinase( PFK)
Fructose-6-P + ATP -----> Fructose-1,6-bisphosphate + ADP
PFK catalyzes 1st committed step in glycolysis (10 steps total)
(Glucose + 2ADP + 2 NAD+ + 2Pi 2pyruvate + 2ATP + 2NADH)

Phosphoenolpyruvate is an allosteric inhibitor of PFK ADP is an allosteric activator of PFK

Allosteric modulators bind to site other than the active site and allosteric enzymes have 4o structure
Fructose-6-P + ATP -----> Fructose-1,6-bisphosphate + ADP

ADP

Allosteric Activator (ADP) binds distal to active site

Vo vs [S] plots give sigmoidal curve for at least one substrate

Binding of allosteric inhibitor or activator does not effect Vmax, but does alter Km

Allosteric enzyme do not follow M-M kinetics

Allosteric T to R transition
ET-I I I ET ER S

ER-S

Concerted model

Sequential model

Covalent modification
Regulation by covalent modification is slower than allosteric regulation Reversible

Require one enzyme for activation and one enzyme for inactivation
Covalent modification freezes enzyme T or R conformation

Phosphorylation /dephosphorylation
most common covalent modification involve protein kinases/phosphatase

PDK inactivated by phosphorylation

Amino acids with OH groups are targets for phosphorylation Phosphates are bulky (-) charged groups which affect conformation

Enzyme Regulation by Association/Disassociation

Acetyl-CoA Carboxylase
acetyl-CoA + CO2 + ATP malonyl-CoA + ADP + Pi 1St committed step in fatty acid biosynthesis In presence of citrate activated In presence of fatty acyl-CoA inactivated citrate polymerized

unpolymerized

Fatty acyl-CoA

Proteolytic cleavage of proenzyme(zymogen)

Proinsulin to Insulin

Blood Clotting
Clotting involves series of zymogen activations Seven clotting factors are serine proteases involved in clotting cascade rxns
X
X X X X X