Topic #4: Protein Function #2--Enzymes pt2

Enzyme Catalyzed Rxns

Enzymes Bind Substrates at Active SIte

Must have these two things

-catalytic site (cofactors, etc.)

Binding sites(

What allows substrates to bind?

Its complementary (types: geometrical, molecular, sterochemical complementarity)

2 Models of Enzyme-Substrate Binding

1) Lock and Key: active site of enzyme perfect match for shape of substrate (rare)

2) Induced Fit Model: Active site is complementary to substrate (doesnt have to be perfect) bc when substrate binds causes shape of enzyme to change

Transition State Formation & Stabilization

• I. Transition state (temporary chemical species formed by collision of reactant
  molecules that ceases to exist as product is formed; possesses high energy &
  extreme instability) formation is rate-determining (rate-limiting) step
  in all chemical reactions

  • A. Activation energy (ΔGact) is amount of free energy required to cause transition state formation

• II. Enzymes work by stabilizing transition state

  • A. Lowers amount of ΔGact needed to form transition state

    • 1. Increases rate of chemical reaction

    • 2. Doesn’t alter chemical equilibrium or thermodynamics of a chem. rxn (cant control if rxn is energy releasing or consuming)

Chem. Rxn Energy Diagram

• y: level of free energy in system

• x-axis: progress of rxn

• red line

  • first take substrate to transition state, necessary energy is activation energy

  • At transition state move past it to make product

  • Rxn that release energy so favorable (exergonic)

• Blue line

  • as energy formed needs stabilizing but doesnt need as much energy and effort so rxn is faster (what catalysts do)

Enzyme Catalytic Mechanisms pt 1 (wont draw)

• I. Proximity & orientation effects on substrates (what all do)

  • A. Bind & immobilize substrates close to each other in proper orientation for chemical reaction to occur

• II. General acid-base catalysis

  • A. Mediated by ionizable groups of AA residues of enzyme

• III. Covalent catalysis (a transient covalent bond is formed between enzyme & substrate)

• IV. Electrostatic catalysis (i.e., ionic interactions)

• V. Preferential binding to transition state (bind greater affinity, stabalizes it more)

Enzyme Catalytic Mechanisms pt 2

• I. Metal ion catalysis (1/3 of all enzymes utilize this mechanism)

  • A. Metalloenzymes (possess tightly-bound transition metal ions to enzyme)

  • B. Metal-activated enzymes (possess loosely-bound alkali (group1) & alkali earth metal ions (group 2)

  • C. Metal ion catalytic mechanisms:

    • 1. Help properly orient substrates

    • 2. Involved in redox reactions

  • 3. Provide electrostatic stabilization & shielding of negative charges (ex., ATP & Mg2+)
    

(ATP only usable when Mg Present)

Enzyme Kinetics

• I. Study of rate of enzyme-catalyzed reactions

  • A. Sheds light on several phenomena:

    • 1. Enzyme affinity determination

    • 2. Enzyme kcat determination

    • 3. Enzyme catalytic mechanism determination

    • 4. Enzyme role in metabolism determination

    • 5. Biochemical & clinical analyses of enzymes

Michaelis-Menten Kinetics (just know what equation is and what tells you)

• I. Michaelis complex (ES) follows steady-state kinetics (i.e., rate of ES breakdown = rate of ES formation) - rxn stays constant (ES constant present and stays constant)

• II. Michaelis-Menten equation is used to quantitate kinetic properties of many
  enzymes
  

Michaelis-Menten Kinetics pt2

• I. Vo: initial velocity (rate before 10% of substrate converted to product)

• II. Vmax: maximum velocity (rate at saturating [S])

• III. Km: Michaelis constant

  • A. Quantitative measure of enzyme affinity for substrate

  • B. [S] at which 50% enzyme molecules are bound to substrate

• 1. [S] = ½ Vmax

• IV. When [S] >>> KM, Vo ≈ Vmax

• V. When [S] = KM, Vo ≈ ½ Vmax
  

(doesnt hold true for all enzymes and can only bind to one substrate)

Catalytically Perfect Enzyme

• I. kcat/KM = specificity constant

  • A. Measure of the catalytic efficiency of an enzyme

  • B. Predictor of enzyme-catalyzed reaction rate when [S] is <<<< KM

• II. Catalytically perfect enzyme has a diffusion-controlled rate (i.e., only rate limiting factor is rate of substrate diffusion to enzyme’s active site)

  • A. Specificity constant of these enzymes = rate of substrate diffusion

  • B. Ex: acetylcholinesterase, carbonic anhydrase, fumarase, triosephosphate isomerase, & beta-lactamase
    

Graphing Michaelis-Menten Kinetic Data

• I. Data obtained from enzyme obeying Michaelis-Menten kinetics will produce a rectangular hyperbolic graph with + slope on an arithmetic scale

  • A. Can be used to infer Vmax & Km of enzyme

-rectangular hyperbolic graph if obey rules (for graph)

• can look for vmax where it plateaus, can also infer km (1/2 of Vmax)

Order KInetics

• I. When [S] <<< KM, Michaelis-Menten enzyme- catalyzed reaction displays 1st-order kinetics

  • A. Rate is directly proportional to [S]

• II. When [S] >>> KM (i.e., when rate approaches Vmax), Michaelis-Menten enzyme-catalyzed reaction displays zero-order kinetics

  • A. Rate is independent of [S]
    

Lineweaver-Burk Plot (used to determine rather than using calculus) (wont calc. know what htey do and interpret them)

• I. Data obtained from enzyme obeying Michaelis-Menten kinetics are graphed on an arithmetic scale as reciprocals (Double Reciprocal Plot)

  • A. Will produce linear graph with + slope

    • 1. Can be used to more easily infer Vmax & KM of enzyme (but not necessarily more accurately)

Substrate Half-Life (t½) pt1

• I. Time required for [S] to be reduced by ½

• II. Can be determined for 1st-order reactions (purple graph), but not for zero order reactions (red graph)

  • A. Rate of a zero-order reaction remains constant (i.e., independent of S])

    • 1. Constant amount of substrate decreases/unit time (e.g., 10 mg/h)

      • i. In this type of chemical reaction, t1/2 is constantly changing

(cant determine half life for 0 order conc. bc always constant amount)

Substrate Half-Life (t½) pt2

• I. Rate of a 1st-order reaction decreases over time (i.e., dependent on [S])

  • A. Constant % of substrate decreases/unit time (e.g., 20%/h)

    • 1. In this type of chemical reaction, t1/2 remains constant

• B. Most physiological processes follow 1st-order kinetics

Temperature-Dependence of Enzyme Activity pt1

• I. In general, rates of chemical reactions are accelerated by increasing temperature & decelerated by decreasing temperature

  • A. The more G‡ required to get a chemical reaction to occur, the more the reaction is temperature-dependent

  • B. Changes in body temperature can positively or negatively affect enzyme activity

Temperature-Dependence of Enzyme Activity pt2

• I. Dramatic increases or decreases in temperature are problematic for living organisms

  • A. Fever (hyperthermia): state of increased body temperature

    • 1. Low-level fever helpful in immune response because it stimulates enzyme activity

    • 2. High-level fever (> 43°C/ 105 F) can cause irreversible enzyme denaturation

• B. Hypothermia: state of decreased body temperature

  • 1. Decreases rate of enzymatic reactions (i.e., decreases metabolic rate of body; loss of vital organ functions occurs)

(temp. dependence based on enzyme activity for graph)

pH-Dependence of Enzyme Activity

• I. Enzymes are pH sensitive

  • A. Because ionization state of catalytic AA is pH- dependent

    • 1. Particularly true for His residue basic R-group

  • B. Because ionization state of AA residues that form intra- & intermolecular interactions necessary for higher order protein structures is pH-dependent

• II. Most enzymes function at physiological pH (7.35 – 7.45)

  • A. This is why acidosis & alkalosis can be fatal

pH-Dependence of Enzyme Activity pt2

• I. Some enzymes function at pH’s below or above normal physiological pH range

  • A. Usually, they are sequestered in specialized areas of cell/body where pH is different than rest of cell/body

    • 1. E.g., acidic enzymes are found in lysosomes & stomach lumen

    • 2. E.g., alkaline enzymes are found in mitochondrial matrix, bile duct, & small Intestine lumen

Inhibitor

• I. Substance that binds to enzyme & temporarily or permanently blocks its catalytic activity

  • A. May interfere with enzyme’s binding of the substrate and/or enzyme’s catalytic action

  • B. In presence of inhibitor, enzyme’s ability to form product is compromised (especially if associated directly)

  • C. Classified based on molecular mechanism

    • 1. Effects are measured kinetically

Competitive Inhibition pt1

1) competitve inhibiton: inhibitor and substrate both going after active site of enzyme as long as inhibitor bound substrate cant bind (gotta have chem features similar to substrate)

Competitive Inhibition pt 2

• I. Competitive inhibitor is structurally like the substrate

• II. Competitive inhibitor binding to active site is reversible (i.e., mediated by noncovalent interactions)

• III. Increasing [S] can overcome competitive inhibitor’s effects

• A. Competitive inhibitors work best at low [S]
  

(only able to bind to free enzyme, binding is reversible ) if more substrate can overcome effects of competitve inhibitors) work best with low substrate conc.

Competitive Inhibition pt 3

• left: mechalis mention graph

  • graph is right shifted & hight is still same

  • tells you competitive inhibitors influence km not vmax

  • raise km no change vmax

• right: line weaver burk plot

  • have same y-int. (1/vmax) but diff. x-int. closer to orgin

  Q: might get 2 graphs asks what type of inhibitor is this

Noncompetitive Inhibiton

• inhibitor not binding to active site but to alosteric site

• can bind to free enzyme or ES (enzyme substrate) complex

• don’t prevent substrate binding, prevent enzyme from catalyzing rxn

NOncompetitive Inhibiotn

• I. Also called mixed inhibition

• II. Noncompetitive inhibitor binding to allosteric site is reversible
  (i.e., mediated by noncovalent interactions)

• III. Increasing [S] does not overcome noncompetitive inhibitor’s effects

Noncompetitive Inhibition

1) michales menton left: km not shift so vmx effected, km not (vmax apparent)

2)line weaver burke (right); y-int. is farther from orgin (lower value) x-int. unchanged (vmax lower km unchanged)

Q:can ask where inhibitor binds and which one it is

Uncompetitive Inhibiton pt1

• inhibitor bind only to ES complex

• reversible binding as long as inhibitor present prevent product formation

Uncompetitive Inhibiton pt2

• I. Uncompetitive inhibitor binding to allosteric site is
  reversible (i.e., mediated by noncovalent interactions)

• II. Increasing [S] makes uncompetitive inhibitor
  more effective
  

Uncompetitive Inhibition pt 3

• left (Menton): slope reduced graph shifted (km value closer to orgin)

  • lowers km & vmax

• Right: parallel lines y-int. further from orgin (value gone down) x-int (further from orgin & value gone down)