Enzymes

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

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

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

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]

Assumptions:

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

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

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

Anabolic/synthetic - create something body needs

Catabolic/degradative - break molecules

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

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

Co-substrates as can bind to active site

Examples - NADH->NAD+

• 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

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)

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 

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

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 

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

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

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

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

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

• 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

• 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

• 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.)

Et = [E]

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

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])

• 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]

• 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))

• 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

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

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

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