Topic 4 - Enzymes Flashcards
What is the relevance of enzymes in medicine?
- Defects in enzymes underlie many diseases
- 30-40% of drug targets are enzymes
- Enzymes can be drugs themselves
- Enxyme activity can be diagnostic/prognostic
- Enzymes are tools in diagnostic/prognostic tests
What are enzymes?
- Biological catalysts of chemical reactions
- Specific action on particular biochemical compounds (substrates)
- Nearly all are proteins
What do enzymes do?
Increase the rate at which the reaction equilibrium is reached, but do NOT shift the position of equilibrium
How do enzymes increase rate of reaction?
- By providing catalytically competent groups for a specific reaction mechanism
- By binding substrates such that their orientation is optimised for the reaction
- By preferentially binding and stabilising the transition state(s) of the reaction
What is the active site of an enzyme?
The region of the enzyme at which substrate binding and conversion to product takes place.
What are the features of an active site?
- 3-dimensional space comprising crucial amino acid residues
- May represent only a small part of the protein structure
- Binds substrate via multiple weak interactions
- Provides substrate specificity because of its unique 3D arrangement of atoms
What is trypsin?
Proteolytic digestive enzyme
Three key amino acids in active site, but far apart in amino acid sequence
Active site is ‘niche’ for binding substrate
What is the equilibrium constant, K?
[A]
What are the three key amino acids making up the active site of trypsin?
They are far apart on the ppt chain:
His57
Asp102
Ser195
What is the michaelis-menten model?
E+S<>ES>E+P
What is the Michaelis constant, Km?
K1
What are the factors affecting enzyme catalysed reaction rates?
- Substrate concentration
- Enzyme concentration
- Temperature
- pH
- Inhibitors
What is a cofactor?
a substance (other than the substrate) whose presence is essential for the activity of an enzyme
What are some major cofactor minerals?
Mg2+ and Ca2+ and trace
elements such as Cu2+, Zn2+, Fe2+/3+, Mn2+ and Mo4+
What is the role of metal ions as cofactors?
- Can be part of active site and/or be involved in electrostatic substrate binding (Zn2+ in carbonic anhydrase, Mg2+ in kinases).
- Can as redox agents (Fe2+/3+ in cytochrome p450s).
- Can regulate activity of enzymes (Ca2+ in calpain).
What cofactor does carbonic anhydrase require?
Zn2+
What is the function of Zn2+ as a cofactor?
Zn2+ in carbonic anhydrase active site binds to H2O and activates it for reaction with CO2
What is the role of NADH and FADH2?
To carry electrons (Recusing power)
What is the role of Coenzyme A?
To carry acetyl units
What is the role of Biotin and Thiamine pyrophosphate?
To carry CO2 units bound to carboxylases
Which water soluble enzyme corresponds to coenzymes FAD and FMN?
Riboflavin (B2)
Which water soluble enzyme corresponds to coenzymes NAD+ and NADP+?
Niacin
Which water soluble enzyme corresponds to coenzyme Thiamine pyrophosphate?
Thiamine (B1)
Which water soluble enzyme corresponds to coenzyme Biotinylated carboxylases?
Biotin
Which water soluble enzyme corresponds to coenzyme cobamide coenzymes?
Cobalamin (B12)
Which water soluble enzyme corresponds to coenzyme tetrahydrofolate?
Folic acid
Which water soluble enzyme corresponds to coenzyme Pyridoxal phosphate?
Pyridoxal (B6)
Which water soluble enzyme corresponds to coenzyme A?
Pantothenic acid
What is pellagra caused by?
Niacin/Tryptophan deficiency
What is the presentation of pellagra?
Dementia, diarrhea and dermatitis
What is the ethanol to acetaldehyde reaction catalysed by?
Alcohol dehydrogenase
What is the acetaldehyde to acetate reaction catalysed by?
Acetaldehyde dehydrogenase
What is reduction?
- gaining of electrons (hydrogen) by one of the atoms
- loss of oxygen
What is Oxidation?
- loss of electrons (hydrogen) by one atom
- gain of oxygen
What can Glucose-6-phosphate dehydrogenase (G6DPH) deficiency cause?
Metabolic defects!
- 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.
What symptoms can G6DPH deficiency cause?
- Haemolytic crisis
- Jaundice
- Can lead to brain damage (kernicterus) in infants
What can trigger a G6DPH deficiency crisis?
- Drugs
- Foods
- Infections
What does the oxidation of G6P by G6PDH generate?
NADPH
What is the role of NADPH?
- Fuel for biosynthesis
- needed to make glutathione, which repairs oxidatively-damaged cell membranes
- RBCs are dependent on G6DPH to make NADPH
What does the pentose phosphate pathway generate?
- Ribose 5-phosphate»> nucleic acids
- NADPH»»Fuel for biosynthesis
What is 6-MP?
- a prodrug that is differentially metabolised
- metabolism by TPMT results in inactivation
What is the role of Thiopurine methyl transferase (TPMT) in adverse response to cancer chemotherapy?
It catalyzes the S-methylation of 6-mercaptopurine (6-MP), azathioprine (AZA), and thioguanine to inactivate these drugs, which are used for treating leukemia and autoimmune diseases. So TPMT deficiency is needed for these drugs to be correctly metabolised.
What are the levels of control of enzymes?
- Inhibition (reversible or irreversible)
- Feedback regulation
- Covalent modification
- Proteolytic activation
What are serine protease inhibitors (serpins)?
a family of proteins that antagonize the activity of serine proteases
What are some examples of serpins?
- Alpha1-antitrypsin (a.k.a. anti-elastase)
- Pancreatic trypsin inhibitor
- Antithrombin III
What does Alpha1-antitrypsin do?
Prevents protease from attacking tissue
What does pancreatic trypsin inhibitor do?
Controls activity of digestive enzyme
What does antithrombin III do?
Switches off blood clotting system
What are some examples of feedback regulation?
- Energy metabolism pathways
- NADP+ on G6PDH
- Purine biosynthesis
- Amino acid biosynthesis
What is protein phosphorylation?
- a universal mechanism of enzyme modulation
- gamma-phosphate of ATP is transferred to amino acid residues
- Enzymes are phosphorylated by other enzymes, called protein kinases
- Phosphate groups are removed by phosphatases
- Charged group induces conformational change in the enzyme
- Key processes in cell signaling by growth factors (hormones, cytokines)
How is zymogen/pro-enzyme activated?
By proteolysis
What is proteolysis?
Irreversible activation of enzyme after removal of part of peptide chain
Where is proteolysis used?
- Digestive enzymes (trypsin, chymotrypsin)
- Blood-clotting system
What does proteolysis require?
tight control by natural inhibitors (for example antitrypsin, antithrombin III)
What does proteolysis prevent?
- enzyme becoming active in the wrong place/wrong time
- eg. premature activation of digestive enzymes in the pancreas
What is chymotrypsin?
- Digestive enzyme
- Mature active protein has 3 polypeptide chains linked by disulfide bonds
- Zymogen precursor is a single polypeptide
What is the connection between chymotrypsin, trypsin and elastase?
evolved from common ancestor
What is trypsin?
- Made as zymogen called trypsinogen
- Removal of amino acids 1-6 cause activation - causes small conformational change
- Initial activation by enteropeptidase, a duodenal enzyme
What is the role of proteases in the blood clotting cascade?
- Massive amplification of initial signal
- Complex interactions in vivo between intrinsic and extrinsic pathways
- Deficiency in any one factor can result in clotting or bleeding disorders
- Many blood Factors are serine proteases
What is antithrombin III?
- A serine protease inhibitor
- Controls clotting by binding tightly to thrombin and serine protease factors
What is the pathway of lysis of blood clots by plasmin?
- TPA adheres to clot and binds plasminogen
- Serine protease activity of TPA cleaves plasminogen to active plasmin
- Plasmin digests fibrin clot to small peptides
What is TPA?
Tissue-type plasminogen activator - plays a role in blood clotting
Which drugs would be administered in coronary thrombosis?
CLOT BUSTERS//thrombolytics
- Recombinant TPA
- Streptokinases (eg. alteplase)
