Enzyme Kinematics and Regulation

3 Different Chemical Reactions in Cells

1) nutrient molecules degraded

2) chemical energy conserved and transformed

3) macromolecules made from simple precursors

Chemical reactions must?

take place at a rate that meets the cell’s needs and must be specific

Specificity of Chem Reactions

a particular reactant should yield a specific product, side reactions producing useless or toxic materials are minimized

Enzyme functions

chemical reactions accelerate and made highly specific, most rxns don’t take place without enzymes

Carbonic Anhydrase

allows for the transport of CO2 from tissues to lungs where it is exhaled

Rate of enhancement by Carbonic Anhydrase

10^7 fold rate of enhancement, each copy of CA converts around 60 CO2 to H2O per second

Carbonic Anhydrate is Added to what 2 reactants?

carbonic acid and bicarbonate

OMP

decarboxylated with half time of 78 million years in neutral aqueous solution at room temp, part of pyrimidine synthesis, OMP to uridine

Proteases

enzymes that convert peptides to carboxyl component and amino component

2 Proteases

trypsin and thrombin

Hydrolysis Site for Trypsin

cleaves the C-terminus of LYS or ARG peptide of basic pair

Hydrolysis Site for Thrombin

more specific than trypsin, cleaves only ARG-GLY blonds with recognition sequence: Leu-Val-Pro-Arg-*-Gly-Ser

Most enzymes are …?

proteins, except for small group of catalytic RNAs,

Catalytic Activity depends on…?

integrity of enzyme’s native conformation and appropriate conditions (pH, temp, salt)

Enzymes Amount

usually present in small amount since they’re not consumed in reactions

Enzyme Activity is…?

regulated

Enzymes require no…?

no chemical groups for activity other than its own AAs, some require cofactors

Temperature and Enzyme Reactions

higher temps accelerate reactions by increasing kinetic energy and collision frequency in catalyzed and uncatalyzed reactions

Very high temps cause…?

denaturation (unfolding) of an enzyme

pH and Enzyme activity

affects ionization rate pf active site and enzyme stability, most have a pH optimum

4 Catalytic Strategies Used by Enzymes

1) covalent catalysis

2) general acid base catalysis

3) metal ion catalysis

4) proximity and orientation

Covalent Catalysis in Enzymes

active site contains reactive group (nucleophile) that is briefly covalently modified

General Acid Base Catalysis in Enzymes

molecule other than water donates or accepts a proton

Metal ion Catalysis in Enzymes

metal ions, + charge, function to stabilize negative charge in reaction intermediate, generate nucleophile by deprotonating water, and bind to S to increase interactions with E increasing binding energy

Example of Metal Ion Catalysis

oxyanion, ammonia acts as base

Proximity and Orientation with Enzymes

enzyme brings two substrates close together and orients the reacting parts of substrate molecule for reaction

6 Classes of Enzymes

1) oxidoreductases

2) transferases

3) hydrolases

4) lyases

5) isomerases

6) ligases

Oxidoreductases

transfer electrons between molecules, catalyze oxidation-reduction reactions

Oxidoreductase Example

Lactate + Lactate Dehydrogenase + Pyruvate

Transferases

transfer functional groups between separate molecules

Transferase Example

serine + serine hydroxy-methyl transferase= glycine

Hydrolases

cleave molecules by the addition of water

Hydrolases Example

urea + H2O and urease = CO2 and NH3

Lyases

break bonds without hydrolysis and oxidation

Lyase Example

Pyruvate + pyruvate decarboxylase= acetaldehyde

Isomerases

move function groups within molecule

Isomerase Example

methylmalonyl CoA + methylmalonyl CoA mutase= succinyl CoA

Ligases

join 2 molecules at the expense of ATP hydrolysis

Ligase Example

Pyruvate + pyruvate carboxylase + ATP= oxaloacetate

Enzyme Cofactors

1) coenzymes- small organic molecules derived from vitamins and can carry electrons of specific functional groups

2) Metals (Mg2+, Mn2+, Zn2+)

Coenzyme 2 Characteristics

1) very tight bind to an enzyme= prosthetic group (heme)

2) can be used by a variety of enzymes, different enzymes using the same cofactors carry out similar chem reactions

Apoenzyme

enzyme minus its cofactor

Holoenzyme

complete catalytically active enzyme plus its cofactor

Least Stable Species in Reaction is…?

the one with the highest free energy, X dagger

Activation Energy

difference in free energy between transition state and the substrate

Where does energy come from for lowering activation energy>

binding energy

Active Site

pocket an enzyme with AA residues that binds the substrate and catalyzes its chemical transformation

Enzyme Substrate Complex Effect

lowers activation energy

6 Interactions between AA side chains and Substrate?

1) Electrostatic

2) hydrophobic

3) hydrogen bonds

4) disulfide

5) Pi-Pi

6) Pi-cation

Good Electrostatic Interactions

between Lys+ and Glu-

Bad Electrostatic Interactions

between Arg+ and Lys+ or Glu- and Asp-

Good Hydrophobic interaction

val and leu

Bad Hydrophobic Interactions

Arg+ and Val or Ser and Phe

Good Hydrogen Bond

between Ser and Glu

Disulfide Bonds

only between Cys and Cys

Pi-Pi bonds

Phe/Tyr and Phe/Tyr

Pi-Cation Bonds

Phe and Lys/Arg

4 Common Features of Active Sites

1) 3D cleft or crevices providing unique environment (water usually excluded unless it’s a reactant)

2) takes up small part of total enzyme volume

3) binds substrates, products, and transition stated by multiple weak interactions

4) structural complementarity after binding

Lock and Key Model of Substrate Binding

shape of active sire modified when S (substrate) binds makes ES complex (enzyme substrate), binding that is too tight makes it harder for S to reach transition state

Induced Fit Model of Substrate Binding

E undergoes change in conformation after binding to S

Effects of Induced Fit Model

permits additional weak binding interactions in transition state, brings specific functional groups in proper position for catalysis

E and S Binding

many weak non-covalent interactions between E and S (H bonds, hydrophobic, ionic interactions)

Weak E and S binding Effects

each weak interaction is accompanied by small release of free energy contributing to stability of interaction

Binding Energy

the total energy derived from ES interactions

Binding Energy Functions

1) major source of free energy used by enzyme to lower activation energy and increase rate of reaction

2) gives enzyme its specificity for a substrate

Weak interactions optimized when…?

the S is in its transition state

Transition State

activated form of a molecule, has undergone partial chem rxn, highest point in rxn coordinate

Enzymes are Complementary to…?

the transition state not the substrate

Enzyme Complementary to Substrate Causes

S fits E too well, would have to break interactions to reach transition state, but a bitter fit to S also means more specificity, less free energy than S alone

Enzyme Complementary to Transition State

1) the S fits to E forming ED, not perfect fit only some interactions

2) S still has to reach high energy transition state to break or change

3) enzyme distorts the substrate to help push it to the transition state

4) when the substrate reaches transition state, the enzyme forms more interactions with it which release binding energy

5) binding energy helps lower activation energy needed to reach the transition state

Lowering Activation energy increases…?

the reaction velocity and more molecules able to get to transition state to be converted to products

Overall Free Energy Change

not usually affected by enzyme, doesn’t change the equilibrium but accelerates the approach to equilibrium by lowering activation energy

What Influences Rates of Enzyme Reactions?

substrate concentration, hyperbolic curves (taper off) in reaction rate vs concentration, evidence of saturation of enzyme by substrate

Cooperative/Allosteric Enzyme Curve

sigmoidal curve (s) shape in respect to the level of substrate

Michaelis-Menten Equation

velocity vs substrate, Km is substrate concentration giving half the max reaction velocity expressed as concentration to describe affinity of enzyme for substrate

Small Km

high affinity for substrate

Large Km

low affinity for substrate

At low concentrations of substrate…?

S<Km and the velocity of rxn is first order, proportional to the substrate concentration (increase in the hyperbola)

At high concentration of substrate…?

S>Km and velocity of reaction is zero order, constant and independent from substrate concentration (taper off in hyperbola)

Why are cellular concentrations of substrates for most enzymes close to Km?

enzymes that operate at their max rate can’t respond to much increase in S concentration and can only respond to large decreases

Lineweaver Burk Plot

when 1/v0 (y) plotted against 1/S (x) it describes straight line with slope of Km/Vmax and y intercept of 1/Vmax, useful to determine Vmax and visualizing effect of enzyme inhibitors

Inhibitor

compound that interacts with enzyme to slow rate of enzyme catalyzed reaction

Reversible Inhibitor

binds to enzymes by noncovalent interactions, enzyme activity recovers when inhibitor is removed

Example of Reversible Inhibitors

ibuprofen and COX (cyclooxygenase) enzyme

Irreversible Inhibitors

react with enzymes through covalent bonds destroys its path, enzyme doesn’t recover when inhibitor is removed

Irreversible Inhibitor Example

aspirin and COX

Reversible Inhibitors can be…?

competitive or noncompetitive

Reversible INhibitors, Competition

reversible binding in active site, inhibitor and substrate compete for enzyme access

Reversible Inhibitor Drug

Statin drug lowers cholesterol by competing against HMG-CoA for active site of HMG CoA reductase, not converted to mevalonate but cholesterol instead

Competitive Inhibitors Effect of Km

increase apparent Km for substrate- higher Km needed to achieve ½ Vmax

Competitive Inhibitor Effect of V max

not affected, inhibition can be reversed by increasing substrate, at high substrate it reaches Vmax

Reversible Inhibitors, Non-Competitive

inhibitor binds reversibly at different site than substrate binding site, changes Vmax but not Km

Non-competitive reversible inhibitor example

Cyanide blocks Cyt C oxidase (complex IV in ETC)

Noncompetitive Inhibition effect of Vmax

lowers vmax, can’t be overcome by increasing substrate

Noncompetitive Inhibition Effect on Km

no effect on substrate binding, Km doesn’t change

3 Types of Irreversible Inhibitors

1) group specific

2) substrate analog

3) suicide inhibitor

Group Specific Irreversible Inhibitor

forms covalent bond with specific AA side chain

Sarin Gas

group specific irreversible inhibitor, inhibits acetylcholinesterase degrades nuerotransmitter ACh allowing muscle to relax after contraction, sarin blocks muscles from relaxing (asphyxiation)

Substrate Analog

looks like natural substrate, will react with enzyme and stay bound

Substrate Analog Example

triose phosphate isomerase (glycolysis)