Enzymes

  • Created by: rosieevie
  • Created on: 13-01-17 18:44

Enzyme Kinetics

Study of enzymes to characterise the rates/steps of catalysis to compare them

Must collect experimentsal data - change in concentration of sub/react over time

Can use a spectrophotometer which measures absorbance change of radiation e.g. visible/UV

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Reaction Rate

In an equilibrium reaction - the reaction appears to halt as conc. remains constant. Rate = Zero

Reaction rate depends on speed of reaction and con. of reactants/substrates

Each reaction has a rate constant (k) 

FORWARD: (Ks->p) x [S] 

BACKWARD (Kp->s) x [P]

Whichever number is higher determines the reaction direction (num. equal at equilibrium)

Enzymes change k by decreasing activation energy (provide an alternative route w/ less energy = more molecules with sufficient energy = more reactions)

Reactions can be 'pushed' in diff. directions by changing concs. Equilibrium ratio will stay the same

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Measuring Reaction Rates

In a reaction [P] can increase over time but not constant rate.

Therefore take zero value and extend = reaction velocity (v mol/min or mol/s)

Also measure enzyme activity (umol/min) or specific activity (umol/min/mg)

Enzyme activity/Total amount of protein = indication of purity

Enzyme-catalysed reactions have two steps:

E + S --binding--> ES ---catalysis--> E + P

Steps may occur at different speeds

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Michaelis-Menten Model 1

(http://biochemistry.wur.nl/CellCycle/MMvergelijking.jpg)

K+2 is called Kcat and K-2 is ignored

K-1/K+1 = [E][S]/[ES] = Dissociation constant (Kd) = Enzymes affinity for [ES]

Small Kd = high affinity [ES]

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Steady-State Kinetics

Assumptions:

  • [ES] constant because reactions occur so quickly
  • [S]>>[E] so [S] is constant - no overall decrease in sub. concentration so rate not effected
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Michaelis-Menten Curve

Vmax - the maximum velocity/rate which an enzyme catalyses a reaction (when all enzymes saturated)

(http://pharmafactz.com/wp/wp-content/uploads/2014/11/michaelis-menten-model-graph.jpg)

  • Helps to work out Km 
  • Km = Vmax/2
  • The line will never reach Vmaz as the sub. would no longer dissolve in solution first
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Types of Reaction

Anabolic/synthetic - create something body needs

Catabolic/degradative - break molecules

Interconverstion - reveresible reaction where same enzyme is used. Substrate and product in equilibrium - balance

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Coenzymes

Organic molcules which provide/remove groups e.g. H+ ions. 

Co-substrates as can bind to active site

Examples - NADH->NAD+

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Enzyme Classification

  • Most names end in -ase
  • Some have common names e.g. trypsin
  • Named for reaction or substrate:
    • Kinase - transfer (PO4)3- from ATP to OH groups
    • Phosphatase - remove (PO4)3- to leave OH (opposite of kinase)
    • Dehydrogenases - remove H with NAD+
    • Glycogen synthase - makes glycogen
  • Every enzyme has unique enzyme commission number (4 digits)
  • 6 enzyme classes (refer to reaction):
    • Oxidoreductases - transfer electrons (H/H-)
    • Transferases - transfer chemical groups
    • Hydrolases - break bonds with water
    • Lyases - reactions involve double bonds
    • Isomerases - transfer groups within a molecule (rearranges bonds)
    • Ligases - formation of bonds using ATP
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Lysosomes

Destroy and recycle cells

Acid sensitive, require low pH - protects rest of cell from digestion

Acid phosphate marker enzyme for lysosomes (tell you are looking at a lysosome)

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Advantages

Maintain pace of life and maintain required conditions for life e.g. pH, body temp

  • Reusable - save resources
  • Specific 
  • Efficient - 100% yield
  • Controllable - start/inhibit reactions 
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The Active Site

Small part of enzyme - rest just holds active site in place

In 3D arrangement the active site is scattered

Contains binding and catalytic residues - source of substrate and reaction specificity

Small part of substrate enters active site - sometimes means not so specific e.g. medicines

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Reaction Specificity

Determined by:

  • 3D residue arrangement - close enough to right bond
  • Chemical properties of residues e.g. positive group only does certain things

Stereospecificity - Optical Isomers

  • Catalytic triad - only 3 amino acids perform catalysis
  • Active site has 3 recognition points
  • If there are stereoisomers only one form of the amino acid will be recognised

Active site also can contain:

  • Metal cofactors e.g. Mg2+, Zn2+
  • Prosthetic groups - organic groups required for overall function e.g. haem 
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Substrate Specificity

Substrate specificity is affected by siz/shape filler and binding affinity

Lock and Key (Fischer)

  • Shape of active site complementary to substrate
  • If not, no enzyme-substrate complex

Induced Fit (Koschland)

  • Mututal conformational change of substrate and enzyme
  • Pull together as substrate enters active site due to bonds
  • Due to any type of bond e.g. ionic, van der Waals, hydrophobic
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What are Enzymes?

Enzyme - one or more polypeptide chain forming a catalytic active site

Substrate - molecule which binds to the active site and undergoes a chemical reaction

Product - result of enzyme action

Enzyme roles:

  • Digestion (pepsin)
  • Blood clotting (thrombin)
  • Control blood pressure (ACE)
  • Defence (lysosyme)
  • Breakdown of toxins (cytochrome)
  • Routine cell processes
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Lineweaver-Burk Plot

Used to calculate Vmax using recipricols - turns plots into a straight line

Vmax - the maximum possible rate (when all enzymes have active sites filled)

Vmaz = Kcat x [E] ---> Kcat = Vmax/[E]            use Vmax to determine Kcat

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Km

Km:

  • [S] which gives half maximum rate
  • [S] at which half of the enzymes have formed enzyme substrate complexes (allows max. flexibility to change reaction rate)

A low Km suggests a high affinity for the substrate

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Biological Significances

Vmax

  • Not signifiicate - need an infinate sub. conc. to retain this
  • Usually low - saturating [S] is unusual
  • Except when drinking excessively

Km

  • Usually high - [S] in cell often close to Km 
  • Each substrate has a different Km for the same enzyme
  • Methanol posioning - treated with ethanol as enzyme has a high affinity for it
  • Determines how active an enzyme is at a particular concentration
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Comparing Enzymes

  • Turnover number - catalytic rate constant (Kcat) - number of reactions per second
  • Enzyme efficiency - catalytic speed if Kcat/Km > 1x108 V is limited by diffusion of substrate not enzyme itself (kinetic perfection)
  • Enzyme potency - how many times faster reaction is with enzyme
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Sigmoidal Curves

  • Suggests a multiple subunit enzyme with co-operative changes in substrate affinity between subunits
  • Substrate binding to one site increases affinity at another
  • Concentration directly regulates enzyme activity
  • Advantage because controllable
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Controlling Enzyme Activity

  • Changing temperature on V - Increase increases V up until denaturing
  • Changing pH on V - Indi. enzymes have optimum pH 
    • Slight drop - enzyme losing positive/negative charges
    • Big drop - denaturing
  • Changing [E] on V - switching genes on and off changes concentration
    • How you get over enzyme saturation (only applies to a fixed conc.)(http://elte.prompt.hu/sites/default/files/tananyagok/IntroductionToPracticalBiochemistry/images/m78f29940.jpg)

Et = [E]

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Direct Enzyme Regulation

Covalent

Most digestive enzymes synthesised in inactive form (damaging) - activated by peptide chain cleavage (irreversible reaction) = ZYMOGENS

Phosphorylisation (reversible) - addition of phosphate group by kinase which distorts active site, denaturing it

Non-Covalent - Allosteric Enzymes

Reversible binding of molecules to specific sites NOT active site -> increase/decrease activity

  • K-type enzyme - effects binding and changes Km
    • Extra polypeptide chain where regulatory molecule reversibly binds
    • Allosteric activator - higher conc leads to increased activity
    • Allosteric inhibitor - higher conc leads to decreased activity
  • V-type regulation - effects catalysis and changes Vmax
    • Change ability of enzyme to catalyse reaction
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Competitive Inhibitors

Enzyme inhibitors reduce enzyme activity - either reversible or irreversible

  • Prevents entry of substrate
  • Binds in active site  or away from active site
  • Lowers V
  • Vmax same (increase [S] = less effect of inhibitor) - delayed reaction though
  • Km larger = weaker affinity
  • Effect similar to less substrate due to competition
  • V = Vmax x [S]/((Km x If) + [S])

(http://pharmafactz.com/wp/wp-content/uploads/2014/11/competitive-inhibitor-graph.jpg)      (http://sites.saschina.org/vincentpx2016/files/2014/09/Image18-qy80dc.jpg)

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Non-Competitive Inhibitors

  • Reaction can never occur even though substrate can bind
  • Binds away from active site
  • Lowers V
  • Smaller Vmax - never reach same
  • Same Km - binding affinity unchanged
  • Same effect as enzyme conc. decreased
  • V = ((Vmax/If) x [S])/Km + [S]

(http://oregonstate.edu/instruct/bb450/summer14/stryer7/8/figure_08_21.jpg)(http://spsphil17.files.wordpress.com/2013/03/asset13.gif)

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Uncompetitive Inhibitors

  • Bind once substrate is in active site
  • Locks substrate in active site but no reaction
  • Vmax is smaller
  • Km is smaller
  • V = Vmax x [S]/(Km + ([S] x If))

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Using Inhibitors as Lab Tools

  • Enzyme structure - inhibitors can stabalise structure and crystalise 'active' form
  • Enzyme purification - coat affinity column beads with inhibitors = only enzyme sticks
  • Active site investigation 
    • Pseudosubstrates bind and irreversibly alter active site
    • Protective masks - determine where active site is
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Enzyme Binding Methods

Difficult to explain kinetics with multiple substrate and product reactions - sometimes order needed or random

Sequential Method - both have to bind before reaction occurs

E + A + B --> EAB ---> ECD ---> E + C + D

  • Order - smaller substrate may have to bind first
  • Random - either can bind first

Ping-Pong Method - substrates never together in the active site

E + A --> EA ---> EC --C leaves --> E' ---> E'B ----> E'D ---> E + D

Covalent bonds to E formed during process

Transition state must form before reaction occurs - can take time. Binding steps partially offset activation energy

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Enzyme Strategies to Increase Reaction Rate

General Strategies

  • Position reactions into correct orientation for interaction
  • Distort reactans making bonds less stable
  • Stabilises transition state - prefered binding (stronger interactions)
  • Chnages environment to favour reaction e.g. pH, hydrophobic, salinity

Best inhibitor drugs resemble transition state 

Specific Chemical Strategies

  • Covalent catalysis - active site residue reacts with substrate
  • Acid-base catalysis - active site residues accept/donate H+ ions
  • Metal ion catalysis - concentrated positive charges

Combo of the 2 which lowers overall reaction energy

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EXAMPLE - PROTEOLYSIS (protein breakdown)

Proteases break down stable peptide bonds efficiently

4 main types focused (all have different substrates and different specificity pockets):

  • Trypsin - large hydrophobic e.g. Phenylaline
    • Negative Coo- in pocket attracts positively charged side chains
  • Chymotrypsin - Lysine, arganine
    • Large pocket lined with hydrophobic residues
  • Elastase - small neutral alanine, serine
    • Only small side chains can enter
  • Thrombin - arganine, glycine

All are initially inactive

Aspartate, Histidine and Serine (catalytic triad) are positioned to create a cleavage site - forms a nucleophile. Undergoes acid/base catlysis and covalent catalysis

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Protease Reaction Mechanism

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