Module 5

Intro

• Life depends on efficiency, selective catalyst chemical reactions

• Most biomolecules are stable w rates of uncatalyzed transformations that are to slow

• Enzymes accelerate, regulate and coordinate these reactions

• Have catalytic power and specificity

• No side reactions

• Can be sensors

Vitalism

• Originally believed that reactions were inseparable from life

• Belief that living things are fundamentally different from non living things in that they contain some non-physical element and are governed by different principles that inanimate objects

  • Ie magic, god, etc

  • Biology and chemistry couldn't explain

• Had many and famous supporter scientists

  • Ie Louis Pasteur

• Eduard Buchner demo that dead yeast still converted sugars into alcohol indicating reactions of life were separate from life

  • Enzyme = in yeast

Co enzymes and co factors

• All enzymes are proteins but not all proteins are enzymes!

• Proteins are complex 3D structures that enable binding of substrates

• Some enzymes can be only protein components and is fully active

• Some enzymes require co factors (inorganic ions) or co enzymes (complex organic molecules or vitamins) for activity

  • Vitamins are required for processes but not produced by the body

  • Ie collagen needs vit C

• Prosthetic group = co enzyme or co factor that is tightly associated to enzyme (degree of association)

• Different enzymes that use same coenzyme usually have similar types of reactions

• Apoenzyme = inactive

• Co factor= activator

• Holoenzyme = active

• Apoenzyme + cofactor = holoenzyme

Catalysts

• Lower amount of energy required for a reaction to proceed

• Sped up equilibrium but don’t change equilibrium

• Unchanged by reaction and recycled to do more

Enzymes vs. chemical catalysts

Circie Effect

• Describes the effect of an enzyme pulling substrates towards them

  • Ie using electrostatic reactions (+ and - charges)

  • Not a random collision

• Enzyme rates of catalysis can approach the physical limit or rates of diffusion of molecules in solution

• Some enzymes have rate determining steps that are as fast as binding of substrates to enzymes

• Some enzymes can catalyze a reaction faster than predicted by diffusion control limits

Equilibrium

• Enzymes catalyze interconversion of substrate and product

• E+S=ES=E+P

  • Both from substrate to product and produce to substrate (both directions)

• Substrate (S) = molecule acted upon by enzyme

• Product (P)= molecule produced by enzyme

• Active site = portion of enzyme responsible for binding substrate to formation of enzyme-substrate complex

Active Site

1. Active site is 3D cleft formed from different parts of polypeptide chains

2. Represents small part of enzyme

3. Unique microenvironments

4. Bound to enzymes by multiple weak interactions

5. Specificity of substrate depends on defined arrangement of atoms in active site

   1. Induced fit and conformation selection caused by substrate binding

   2. Enzymes change active sites (flexible) to bind to substrate = both enzyme and substrate change conformation

Enzyme Specificity

• Lock and key - no change in the enzyme or substrate (not accurate)

• Hand in glove - change in both enzyme and substrate (accurate)

Free energy

1. Reaction is spontaneous only if delta G is neg

   1. Spontaneous means reaction will occur without input of energy and it releases energy (exergonic)

2. Reaction cannot take place spontaneously if delta G is positive

   1. Needs input of free energy to drive reactions (endergonic)

3. If system is at equilibrium there is no net change in concentration of the products and reactants and delta G is 0

4. Delta G of a reaction depends only on free energy of product minus energy of reactants (delta G is independent of steps of transformation

5. Delta G provides no info  about rate of reaction

• Activation energy (delta G) between S and P determines rate at which equilibrium is reached

• Enzymes provide lower energy pathway between substrate and product lowering delta G (activation energy of the transition state and increasing rate of reaction)

• Rate of reaction and activation energy is inverse and exponential relationship

• Difference in free energy between S and P determines equilibrium of reaction

• Enzymes do not influence the difference in free energy and don’t influence equilibrium

Catalysis

• Forces lower the activation energy

• Chemical and binding effects

1. Binding effects (physical interactions)

   1. Substrate binding

      • Reducing entropy

      • Alignment of reactive functional groups of enzyme with substrate

      • Desolation of substrate (removal of water molecules) to expose reactive groups

      • Distortion of substrates

      • Induced fit of enzyme in response to substrate binding

   2. Transition state stabilization

      • Increased interaction of enzyme and substrate in transition state

      • Enzyme distorts substrate forcing it toward transition state

      • Active site is complementary to transition state in shape and chemical character

      • Enzymes bind transition states 10 power of 10-15 times more tightly than their substrates

      • Active site must be similar enough to substrate to ensure specificity, different enough to promote change

   • Binding of substrate in active site provides specificity and catalytic power

   • Limited to binding properties can still increase reaction rate by over 10,000 folds

   • E+S=ES=ETS=E+P

     • Substrate goes to E-ES

     • Transition state goes to ES-ETS

       • Overlap of effects

   • Transition state analogs - competitive inhibitors

     • Stable compounds that resemble unstable transition states

     • Potential therapeutic applications as competitive inhibitors (molecules that bind to active site of enzyme and tend to resemble substrate molecule)

     • TSAs can bind to active site of target enzyme with high affinity to prevent substrate binding

     • Transition states are short lived and unstable

2. Chemical effects

• After substrate binding enzyme can act upon substrate to promote formation of product

• Active site usually contains chemically reactive side chains

  • Polar, ionizable side chains (triprotics) ie Asp, Glu, His, Cys, Tyr, Lys, Arg, Ser

  1. Acid base catalysis

     1. Reactive acceleration achieved by catalytic transfer of proton

     2. Side chains of aa act as base or acids

     3. His with pKa near physiological pH often involved

     4. pKa of functional groups influenced by chemical microenvironment

  2. Covalent catalysis

     1. Part of enzyme mechanism the substrate is covalently bound to enzyme to form a reactive intermediate

        1. Ex. A-X + E = X+E + A STAGE 1 (form covalent linkage to enzyme)

        2. X-E + B = B=X + E STAGE 2 (regenerate the free enzyme)

        3. Ex. Sucrose phosphorylase:

           1. Step 1: Glucosyl residue is transferred to enzyme

           2. Glucose is transferred to phosphate

Enzyme kinetics

• Kinetics = study of velocity of reactions

  • Substrate= product

• Velocity of a reaction quantified as change in conc of product over time

  • V=change in P/ change in time

• Units of concentration over time (ex. Mmoles/sec or moles/min

• Enzymes are proteins that can be influenced by many factors which influence structure and activity

  • Temp and pH sensitive

  • Different enzymes have different optimum temps and pHs

  • Most cells are at physiological levels ( 37 degrees C and ph of 7.4) which is optimum in most places but not all

    • Ie lysosomes break things into building blocks using enzymes, if they burst into the contents of the cell the enzymes wont function at the pH inside cell 

• Velocities influenced by enzyme and substrate concentration

  • Look at velocity vs. substrate conc

• Velocity = change in product concentration over time (need to measure product formation before equilibrium reached)

• Initial velocity = velocity at beginning of enzyme catalyzed reaction prior to product accumulation

• k1 and k-1 = rapid non covalent interactions between enzyme and substrate

• k2 = rate constant of formation of product from ES

  • V0= [ES]k2

Michaelis Menton Kinetics - Steady state assumption

• Rate of formation of ES complex = rate of breakdown

  • [E][S]k1 = [ES]k-1 +[ES]k2

    • Rate of formation left

    • Rate of breakdown right

• Relationship between substrate concentration and initial velocity

  • Km = concentration of substrate required to reach 1/2 Vmax

    • Concentration of substrate required for enzyme to function at half max velocity

    • In vivo

    • Most enzymes function at half max velocity

  • Vmax= max velocity of enzyme

    • V0=Vmax[S]/km+[S]

• S<Km, enzymes sensitive to changes in substrate concentration but have little activity (bottom of graph)

• S>Km, enzymes have high sensitivity but are insensitive to changes in substrate concentration (top of graph)

• When S= Km, enzyme has significant activity and is responsive to changes in substrate concentration (ideal)

• Vmax is independent of substrate concentration (all  enzymes are full)

• Km = amount of substrate required to get to 1/2 max velocity

  • Substrate concentration NOT velocity

• Ex. Velocity of a reaction when substrate concentration is equal to Km

  • V0= 1/2 Vmax

• Ex. Velocity when substrate concentration is double Km

  • V0=2/3 Vmax

• Velocity when substrate concentration is a third of Km

  • Vo= 1/4 Vmax

Lineweaver Burk Plots

• Describe relationship between S and V0

• Double reciprocal plot 1/V0 and 1/S

• More precise method

• Used to determine Vmax and Km

  • 1/V0 = Km/Vmax[S] + a/Vmax

  • To find Vmax reciprocal the value

  • Find

  •  negative reciprocal value

Enzyme turnover number

• kcat

• Equals number of molecules of substrate converted to product per unit time under saturating conditions

• Vmax/[Et]

Reversible Enzyme inhibition

• Inhibitor = compound that binds to enzyme to interfere with its activity

• Prevent formation of ES or breakdown to E and P

• Reversible inhibitors bind to enzyme by non covalent interactions

  • Competitive

  • Uncompetitive

  • Noncompetitive

• Competitive inhibitors

  • Resemble substrate and compete with substrate for binding the active site

  • Ie Antibiotic sulfanilamide is competitive inhibitor for bacterial enzyme that has PABA as substrate

  • Bind only free enzymes

  • Effect of competitive inhibitors can be overcome with excess of substrate (washing out)

  • Vmax is same but apparent Km is increased

  • Crosses same place on y axis

• Uncompetitive inhibitors

  • Bind to only ES complex

  • Vmax is decreased by conversion of ES to ESI which cannot form product

  • Reduce ES

  • Decrease in Km

  • Ie Roundup

• Noncompetitive inhibitors

  • Binds to E and ES

  • Vmax is decreased with no change in Km

  • Don’t influence S binding

  • Reduces number of active enzyme molecules

  • Ie antibiotic doxycycline is non competitive inhibitor of bacterial enzyme (collagenase)

• Recap:

  • Competitive: Binds E and increases Km

  • Uncompetitive:  Binds ES and decreases both Vmax and Km

  • Noncompetitive: binds E or ES and decreases Vmax

Serine Proteases

• Digestive enzymes that cleave peptide bonds in protein structures

• Share similar sequences and active site residues

• Synthesized and stored in pancreas as inactive zymogens to prevent damage to cellular proteins

  • Zymogens are activated at appropriate time by selective proteolysis

• Has covalent and acid base catalysis

• Specifics hat reflect unique substrate binding pockets:

  • Thrombin cleaves Arg-Gly  bonds

  • Trypsin cleaves Lys and Arg bonds (+)

  • Chymotrypsin cleaves Phe, Tyr or Met (aromatics)

  • Elastase cleaves Gly and Ala (small hydrophobics)

  • Papain cuts all peptide bonds

• Have conserved catalytic mechanisms based on catalytic triad of residues (Asp D, His H and Ser S)

  • His acts to accept and donate proton at each of 2 stages of the reaction mechanism

  • Asp stabilizes the positively charged His to facilitate serine ionization

  • Ser attacks carbonyl group of peptide bond to be cleaves (covalent catalysis)

• Mechanism

  • Phase 1:

    1. Acid base - His acts as a base to extract proton from hydroxyl of Ser, Activates oxygen of hydroxyl group.

    2. Covalent - formation of covalent linkage from hydroxyl group of Ser to carbonyl carbon of peptide bonds to be cleaved in substrate

    3. Acid base - His acts as acid to donate proton to amin group of peptide bond to be cleaved, cuts substrate peptide into 2 pieces

  • Phase 2:

    1. Acid base - His acts as base to extract proton from water molecule, activating oxygen

    2. Covalent - activated water molecule attacks point of covalent linkage between enzyme and substrate

    3. Acid base - His acts as acid to donate proton to reform hydroxyl group of Ser

Regulation of enzyme activity

• Enzymes are regulated by controlling amount of enzyme (long term) or adjusting activity of constant quantity of enzyme (short term)

1. Regulation of enzyme availability

   1. Location, rates of synthesis and degradation

2. Regulation of enzyme activity

   1. Covalent modification

      • Phosphorylation, methylation, glycosylation

   2. Non covalent modification (allosteric)

      • Allosteric regulation

• Pathways controlled by negative feedback inhibition by final product pathway

• Final product inhibits enzyme catalyzing first unique and committed step

  • Conserves material and energy and prevents accumulation of intermediates

• F end product needed in limited amounts and cannot be stored

• A valuable and showed be conserved unless F is needed

• B, C, D and E no biological role - only intermediates in production of F

• Branched pathway occurs by final product of each branch acting to inhibit enzyme catalyzing 1st unique committed step of branch

• 2 pathways cooperate to form a single product

• Molecules before merger can inhibit first step of their branch and activate first step of opposing branch

Allosteric enzymes

• Info sensors to coordinate cellular metabolism

• Regulated by interactions w metabolic intermediates

• Bind non covalently at other sites than active sites

• Quaternary structure

• Branch point reactions

• Slow = rate limiting step of pathway (so it can speed up pathway)

• Don’t obey Michaelis Menton kinetics - have sigmoidal curves

Properties

• Changed by inhibitors and activators (modulators)

• Bind non covalently to enzymes

• Rapid transition between active (R) and inactive (T) conformations

• Substrates and activators bind to only R state

• Inhibitors bind to T state

  • Substrate binding disrupts R to T equilibrium in favor of R

  • Cooperative activation of allosteric enzymes

• Can transition from less active to more active state within narrow range of substrate conc

• Sensitive to changes in substrate conc near Km

• Threshold effect below certain substrate conc little enzyme activity

  • After threshold, enzyme activity increases rapidly (on/off)

Ex. Phosphofructokinase 1

• PFK1 catalyzes early step of glycolysis (energy production)

• PEP is intermediate near end of pathway is allosteric inhibitor of PFK1

• ADP is allosteric activator of PDK1

• Ratio of PEP/ADP high = PFK1 inhibited

• Ration PEP/ADP low= PFK1 activated + glycolysis produces more ATP from ADP

  • Conc of PEP and ADP act allosterically through RFK1 to regulate activity of entire pathway

• PFK1 responsive to conc of substrate + modulators

• Constant levels of substrate activity of enzyme can be modulated through changes in levels of allosteric modulators

Covalent modification

• Modifying group to change aspect of protein behavior

  • Methylation, acetylation

• Most common is phosphorylation

• Reversible w one enzyme catalyzing the addition of group and other enzyme catalyzing removal

• Kinase add phosphoryl groups, phosphates remove them

• Affects Serine, Tyrosine and Threonine

 Glycogen Metabolism

• Production and utilization of glycogen is controlled by 2 enzymes

  • Glycogen synthase (anabolic) = produce glycogen from glucose

  • Glycogen phosphorylase (catabolic) = breakdown of glycogen into glucose

• Don’t want both at same time

• Hungry release glucagon hormone released

• Scared release epinephrine released

  • Both enzymes are phosphorylated

• Activated catabolic enzyme and inactivates anabolic enzymes

• Favors breakdown of glycogen into glucose

• Hormone in fed state insulin both enzymes are unphosphorylated

• Anabolic enzyme is active and catabolic is inactive

• Favors storage of glucose within glycogen