Enzymes

1
Enzymes
Crystal structure of the complex of
phosphofructokinase from Escherichia coli with
its reaction products. From Protein Data Bank
PDB ID 1PFK Shirakihara, Y., Evans, P. R.
Crystal structure of the complex of
phosphofructokinase from Escherichia coli with
its reaction products. J Mol Biol 204 pp. 973
(1988)
2
Functions

• Enzymes are proteins which
• catalyse reactions so that they proceed at a
  useful rate
• channel substances into specific pathways
• can be regulated to control flux through a
  pathway

3
Catalysis

• Catalysts increase reaction velocity without
  themselves being changed
• Can accelerate a reaction in both directions
• Some enzymes non-equilibrium
• Do not affect the state of equilibrium of a
  reaction
• simply allow equilibrium to be reached faster

4
Activation energy

• Molecules must be activated before they can
  undergo reaction
• Reactants must absorb enough energy from
  surroundings to destabilise chemical bonds
  (Energy of activation)
• Transition state
• Intermediate stage in reaction where reactant
  molecule strained or distorted but reaction not
  yet occurred

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.346.
5
Activation energy

• A catalyst lowers the energy of activation
  required by
• Forcing molecules into conformation that favours
  reaction
• ie may reorientate molecules
• Change in free energy is the same per reaction

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.348.
6
Activation energy

• Sometimes catalysts cause one large energy
  barrier to be replaced by two smaller ones
• Reaction passes through intermediate stage

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.349.
7
Active site

• Enzyme binds substrate(s) at active site
• Complexity of tertiary structure (and therefore
  active site) confers high specificity
• Pocket or cleft surrounded by amino acid side
  chains that aid catalytic process

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.355.
8
Induced fit model

• Binding of substrate(s) at active site
• Causes distortion of enzyme and substrate
  (induced fit)
• puts substrate into transition state
• Better orients substrate(s) for reaction
• Directly promotes catalytic event
• Amino acid side chains or ions may be positioned
  to aid in catalysis

From Matthews, CK van Holde KE (1990)
Biochemistry. Redwood CityBenjamin Cummings
p.351.
9
Michaelis-Menten Kinetics

• Vmax approached at high substrate concentrations
• Enzymes with tertiary structure display Michaelis
  Menten kinetics
• Km substrate concentration at 0.5 Vmax
• Measure of relative affinity of enzyme for
  substrate

From Elliott, WH. Elliott, DC. (1997)
Biochemistry and Molecular Biology. Oxford
Oxford University Press. p162
10
Allosteric enzymes

• Enzymes with quaternary structure display
  sigmoidal binding kinetics
• Have more than one active site
• K0.5 substrate concentration at 0.5 Vmax
• Regulate substrate concentrations at very stable
  levels (usually close to K0.5)

From Elliott, WH. Elliott, DC. (1997)
Biochemistry and Molecular Biology. Oxford
Oxford University Press. p163
11
Regulation of enzyme function

• Enzyme activity regulated in two ways
• Change amount of enzyme
• Change rate of catalysis (specific activity)

12
Regulation of enzyme function - changing levels
of enzymes

• Enzyme levels manipulated by controlling rates of
  turnover
• Different enzymes have different half-lives
• More rapidly degraded enzymes occupy key points
  in pathways
• Allows more rapid control of enzyme levels
• Changes in enzyme levels usually result in slow
  responses of metabolic control

13
Regulation of enzyme function - regulation of
specific activity

• Specific activity regulated in two ways
• Allosteric control
• Ligands bind to site other than active site
• Ligand may be substrate, product or other
  molecule
• Positive modifier increases activity at given S
• Negative modifier decreases activity at given S
• Enzyme can have allosteric sites for gt1 effector
• Allows control from number of metabolic areas
• Specific activity is net result of action of all
  effectors

From Elliott, WH. Elliott, DC. (1997)
Biochemistry and Molecular Biology. Oxford
Oxford University Press. p163
14
Regulation of enzyme function - regulation of
specific activity

• Covalent modification
• Some enzymes inactive until covalently modified
• Inefficient to be active all of time
• Usual modification is phosphorylation by a kinase

15
Covalent modification - Example

• Glycogen phosphorylase
• Converts glycogen to glucose-1-phosphate
• Phosphorylase kinase activates Gphos via
  phosphorylation
• Phosphorylase kinase activated by another kinase
  PKA
• PKA activated by cAMP
• cAMP produced as result of Epinephrine release at
  start of exercise
• Metabolic cascade amplifies original signal

From Elliott, WH. Elliott, DC. (1997)
Biochemistry and Molecular Biology. Oxford
Oxford University Press. p173
16
Regulation of enzyme function - inhibition

• Competitive inhibitors
• Mimic substrate and compete for active site
• Non-competitive inhibitors
• Allosteric regulators
• Covalent modifiers
• If covalent modification irreversible usually
  toxic

17
Regulation of enzyme function - feedback control

• Specific regulatory enzymes strategically placed
  and regulated by feedback mechanisms
• Regulatory enzymes usually non-equilibrium
• First enzyme in pathway usually strategic place
  for control

From Elliott, WH. Elliott, DC. (1997)
Biochemistry and Molecular Biology. Oxford
Oxford University Press. p162
18
Co-enzymes

• Range of protein side chains not sufficient for
  all enzyme functions
• Coenzymes assist enzymes
• Prosthetic group
• Vitamins important for incorporation into
  coenzyme structures
• eg NAD contains niacin