ENZYMES
INTRODUCTION TO ENZYMES
Relevance of enzymology to medicine
Drug action, biochemical defects in enzymes underlie disease, clinical diagnosis/prognosis
Many drugs are enzyme inhibitors, e.g. codeine
How do enzymes decrease the activation energy of a reaction
By providing catalytically competent groups (e.g. metal ions) for a specific reaction mechanism
By binding substrates such that their orientation is optimized for the reaction
By preferentially binding and stabilising transition states of the substrate
Increase the rate at which the reaction equilibrium is reached, but do not shift the position of equilibrium
Active site
ESC EPC takes place
It’s a 3D entity comprising crucial amino acid residue
Binds substrate via multiple weak interactions
Provides specificity because of its unique conformation of atoms
Assay: A procedure for measuring the biochemical or immunological activity of a sample
Enzyme kinetics
k1 is the rate of formation of enzyme-substrate complex
k2 and k3 are the rates of dissociation of ES complex
Km: substrate conc at which the rate is half Vmax ; a low Km indicates high affinity of the enzyme for its substrate- low
conc of substrate required to saturate enzyme
The Michaelis-Menten Equation
Km will always be the same
pH
Temperatur
e
Irreversible
inhibition
Covalent modification of amino acid side chains in the active site
E.g. carboxymethylation of cysteine side chains by idoacetamide and snake venom for acetylcholinesterase
Reversible inhibition
Competitive Non-competitive
Enzyme can bind substrate OR inhibitor, but not both Inhibitor and substrate can bind simultaneously
at the same time Binding occurs at independent sites
Substrate and inhibitor compete for active site Inhibitor alters conformation or accessibility of active
Substrate and inhibitor often share similar structures site.
Pharmacologically important Inhibition not affected by high substrate concentration
Inhibition overcome by high substrate concentration
, Aspirin: Covalent modification of a serine residue in the active site. Competitive, IRREVERSIBLE
Ibuprofen: Binds to active site, but not covalently attached. Competitive, REVERSIBLE
Cofactors
Metal ions provided as trace elements in the diet
Essential components in active sites in some enzymes
Cu2+, Zn2+, Fe3+, Mn2+, even Mo4+, occur naturally
Metal ion may bind reactants electrostatically, or may act as oxidising agents
Other metal ions (e.g. Mg2+, Ca2+) may be required for activity, but are not part of
active site.
REGULATION OF ENZYME ACTIVITY
Coenzymes
E.g. water soluble vitamins
They function as carriers of reaction components
o NADH and FADH2 carry electrons (‘reducing power’)
o Coenzyme A carries acyl units
o Biotin and thiamine pyrophosphate carry CO2 units (bound to
carboxylases)
o PO3 group gives specificity between NADP and NAD
Glucose- 6-Phosphate dehydrogenase - most commonest enzyme deficiency
X-linked recessive, most carriers asymptomatic
The enzyme produces a large proportion of the body’s NADPH needed to
drive biosynthesis of nucleic acids, lipids etc.
Where symptomatic, can include haemolytic crises, jaundice (can lead to
brain damage (kernicterus) in infants)
Crises can be triggered by certain drugs, foods or infections
G6PDH mutations can effect either the stability of the dimeric enzyme, the
binding of the regulatory NADP+ cofactor, or the structure of the active site
itself
Extra glucose goes into PPP – pentose phostphate pathway
Why do we need NADPH?
NADPH is very important in maintaining the levels of reduced glutathione in cells
Glutathione keeps cell membranes happy, by reducing lipid hydroperoxides
If they are not removed, the hydroperoxides react to cause cleavage of the acyl chains and ultimately red cell lysis.
Glutathione reduces them to alcohols by glutathione peroxidase
The resultant GSSG is reduced back to GSH by NADPH, catalysed by glutathione reductase
Anti-malarial primaquine can trigger a haemolytic crisis; it stimulates peroxide formation thereby increasing the
demand for NADPH to a level that the mutant enzyme cannot provide
Thiopurine methyl transferase (TPMT)
Thiopurine drugs are used as anti-cancer agents
Can be incorporated into DNA during replication in actively growing cells, but
then block further extension of the DNA chain; growth is arrested
Because cancer cells have a particularly high demand for new DNA chains
(because they grow more rapidly than most other cells), these cytotoxic drugs will
tend to kill cancer cells more effectively than (most) other cells in the body
INTRODUCTION TO ENZYMES
Relevance of enzymology to medicine
Drug action, biochemical defects in enzymes underlie disease, clinical diagnosis/prognosis
Many drugs are enzyme inhibitors, e.g. codeine
How do enzymes decrease the activation energy of a reaction
By providing catalytically competent groups (e.g. metal ions) for a specific reaction mechanism
By binding substrates such that their orientation is optimized for the reaction
By preferentially binding and stabilising transition states of the substrate
Increase the rate at which the reaction equilibrium is reached, but do not shift the position of equilibrium
Active site
ESC EPC takes place
It’s a 3D entity comprising crucial amino acid residue
Binds substrate via multiple weak interactions
Provides specificity because of its unique conformation of atoms
Assay: A procedure for measuring the biochemical or immunological activity of a sample
Enzyme kinetics
k1 is the rate of formation of enzyme-substrate complex
k2 and k3 are the rates of dissociation of ES complex
Km: substrate conc at which the rate is half Vmax ; a low Km indicates high affinity of the enzyme for its substrate- low
conc of substrate required to saturate enzyme
The Michaelis-Menten Equation
Km will always be the same
pH
Temperatur
e
Irreversible
inhibition
Covalent modification of amino acid side chains in the active site
E.g. carboxymethylation of cysteine side chains by idoacetamide and snake venom for acetylcholinesterase
Reversible inhibition
Competitive Non-competitive
Enzyme can bind substrate OR inhibitor, but not both Inhibitor and substrate can bind simultaneously
at the same time Binding occurs at independent sites
Substrate and inhibitor compete for active site Inhibitor alters conformation or accessibility of active
Substrate and inhibitor often share similar structures site.
Pharmacologically important Inhibition not affected by high substrate concentration
Inhibition overcome by high substrate concentration
, Aspirin: Covalent modification of a serine residue in the active site. Competitive, IRREVERSIBLE
Ibuprofen: Binds to active site, but not covalently attached. Competitive, REVERSIBLE
Cofactors
Metal ions provided as trace elements in the diet
Essential components in active sites in some enzymes
Cu2+, Zn2+, Fe3+, Mn2+, even Mo4+, occur naturally
Metal ion may bind reactants electrostatically, or may act as oxidising agents
Other metal ions (e.g. Mg2+, Ca2+) may be required for activity, but are not part of
active site.
REGULATION OF ENZYME ACTIVITY
Coenzymes
E.g. water soluble vitamins
They function as carriers of reaction components
o NADH and FADH2 carry electrons (‘reducing power’)
o Coenzyme A carries acyl units
o Biotin and thiamine pyrophosphate carry CO2 units (bound to
carboxylases)
o PO3 group gives specificity between NADP and NAD
Glucose- 6-Phosphate dehydrogenase - most commonest enzyme deficiency
X-linked recessive, most carriers asymptomatic
The enzyme produces a large proportion of the body’s NADPH needed to
drive biosynthesis of nucleic acids, lipids etc.
Where symptomatic, can include haemolytic crises, jaundice (can lead to
brain damage (kernicterus) in infants)
Crises can be triggered by certain drugs, foods or infections
G6PDH mutations can effect either the stability of the dimeric enzyme, the
binding of the regulatory NADP+ cofactor, or the structure of the active site
itself
Extra glucose goes into PPP – pentose phostphate pathway
Why do we need NADPH?
NADPH is very important in maintaining the levels of reduced glutathione in cells
Glutathione keeps cell membranes happy, by reducing lipid hydroperoxides
If they are not removed, the hydroperoxides react to cause cleavage of the acyl chains and ultimately red cell lysis.
Glutathione reduces them to alcohols by glutathione peroxidase
The resultant GSSG is reduced back to GSH by NADPH, catalysed by glutathione reductase
Anti-malarial primaquine can trigger a haemolytic crisis; it stimulates peroxide formation thereby increasing the
demand for NADPH to a level that the mutant enzyme cannot provide
Thiopurine methyl transferase (TPMT)
Thiopurine drugs are used as anti-cancer agents
Can be incorporated into DNA during replication in actively growing cells, but
then block further extension of the DNA chain; growth is arrested
Because cancer cells have a particularly high demand for new DNA chains
(because they grow more rapidly than most other cells), these cytotoxic drugs will
tend to kill cancer cells more effectively than (most) other cells in the body
