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Enzymes
Lecture 4
Catalytic proteins
Recap...
-  Proteins are the key functional molecules in life
-  Proteins have many structures, resulting in a wide range of functions
-  A proteins specific conformation determines how it functions
Enzymes
•  Are a type of protein that acts as a catalyst-speeding up
chemical reactions
•  A catalyst is defined as a chemical agent that changes
the rate of a reaction without being consumed by the
reaction
•  Enzymes are biological catalysts
Chemical reactions
•  Chemical reactions involve the breaking and forming
of bonds, this requires energy
•  Activation energy is the initial investment of energy
needed to start a reaction
•  In most chemical reactions this is provided in the form
of heat
•  humans need to maintain a temperature of 37˚C
•  Enzymes provide the activation energy for reactions to
proceed at normal temperatures in biological systems
Enzymes
• What enzymes do therefore is: Reactants
Transi6on state
Products (ready to react) +
catalyst
Catalysts act here • Biochemists call ‘reactants’ substrates Substrate Transi6on state
Products (ready to react) Enzymes act here +
enzyme
+ lipase
• Enzymes are substrate-­‐specific – each substrate fits into the enzyme’s ac6ve site Eg. Sucrase
1 Active site is available for
a molecule of substrate, the
reactant on which the enzyme acts.
2 Substrate binds to
enzyme.
Substrate
(sucrose)
(sucrase) Transi6on state (sucrase) Glucose
OH
Fructose
Enzyme
(sucrase)
H 2O
H O
(sucrase) 4 Products are released.
3 Substrate is converted
to products.
Chemical reaction involved:
Hydrolysis of sucrose involves the breaking of the bond between glucose and fructose the breaking of a bond in the water molecule the forming of 2 new bonds How do they work?
•  Complex molecules have the
potential to breakdown
spontaneously
(2nd law of thermodynamics= the
universe tends towards disorder)
•  They continue to exist because the
initial activation energy required to
breakdown can’t be reached to
initiate a breakdown reaction
•  An enzyme lowers the activation
energy required for a reaction
enabling the substrates to absorb
enough energy even at a moderate
temperature to reach transition
state
Enzyme shape
•  Enzymes are substrate specific
–  The substrate fits like a key in a lock
–  When the substrate and enzyme are joined
the catalytic action of the enzyme converts
the substrate to product
•  The specificity of an enzyme for a substrate
results from the shape of the enzyme –due
to a compatible fit between the active site
and the substrate
•  The substrate binds to the “active site” of the
enzyme
-This is the catalytic centre of the enzyme
- The active site is typically a pocket or
groove on the surface of the enzyme
Active site – groove on surface of enzyme
Groove
Eg. Lysozyme- an enzyme that
Breaks down bacterial cell walls
by recognizing and binding to
specific molecules on the bacteria
(a) A ribbon model
Groove
(b) A space-filling model
Enzymes “active site”
•  Is usually formed by only a few amino
acids
•  Is not rigid
•  The side chains of the aa’s in the
enzyme interact with the chemical
groups of the substrate- enzyme
changes shape and fits even better
around the substrate- INDUCED FIT
•  Induced fit brings the chemical groups
of the active site into new positions
enhancing the ability of the enzyme to
catalyse the reaction
•  The rest of the protein structure
provides the structural framework that
determines the configuration of the
active site
Induced fit
Induced fit
6 important Features of enzymes 1.  Side chains of amino acids make up the ac6ve site 2.  Substrates bind via weak interac6ons: hydrogen bonds, hydrophobic interac6ons 3. Enzymes act at op6mal pH and temperatures: Enzyme ac6vity Enzyme ac6vity 37oC 7.4 temp pH Excep6ons: Thermophiles 70⁰C Excep6ons: pH 2 in stomach – pepsin pH 8 in intes6ne -­‐ trypsin Features of enzymes
4. Many require non-protein helpers to aid catalysis
- ‘co-factors’
Eg: calcium, iron.
• 
If the co-factor is organic it is a co-enzyme
- Most vitamins are coenzymes Eg: vitamin C, NAD+
• 
Vitamin C is used by an enzyme to cross-link collagen. Vitamin C
deficiency causes scurvy – a disease of dysfunctional cross-linking
of collagen
• 
NAD+ and Zinc are co-factors used by
alcohol dehydrogenase
Alcohol metabolism
Enzyme 1
Co-factor= zinc
Co-enzyme = NAD+
Enzyme 2
Alcohol dehydrogenase
Acetaldehyde dehydrogenase
ADH Alcohol NAD+ NADH
Co-enzyme
Acetaldehyde + 2x H NAD+ NADH
Co-enzyme
Acetate/ ace6c acid + H poisonous
Blood circulation
Broken down into
carbon and water during
the Krebs cycle
Alcohol metabolism
Enzyme 1
Co-factor= zinc
Co-enzyme = NAD+
Enzyme 2
Alcohol dehydrogenase
Acetaldehyde dehydrogenase
less efficient
Very efficient
ADH Alcohol NAD+ NADH
Co-enzyme
Methanol
Acetaldehyde + 2x H formaldehyde
NAD+ NADH
Acetate/ ace6c acid + H Co-enzyme
poisonous
Blood circulation
• Women have less ADH in their stomach than men
Broken down into
carbon and water during
the Krebs cycle
• “Flush syndrome”:
apparent in many east asians and american indians
symptoms: headaches, nausea, vomiting, heart palpitations
50% of individuals from japenese descent show flush syndrome
Features of enzymes
5. Many drugs inhibit enzymes
•  Drugs can compete with
the substrate and prevent
the reaction happening
HMG-­‐CoA Mevalonic acid Cholesterol ENZYME: HMG-­‐CoA reductase Eg. Statins inhibit HMG-CoA
Reductase, a key enzyme
in cholesterol synthesis
HMG-­‐CoA – statins therefore lower
cholesterol and protect
or against heart disease.
Lovostatin competes with
the substrate for the
active site
lovosta6n HMG-­‐CoA reductase Features of enzymes
5. Many drugs inhibit enzymes
•  Drugs can compete with
the substrate and prevent
the reaction happening
HMG-­‐CoA Mevalonic acid Cholesterol ENZYME: HMG-­‐CoA reductase Eg. Statins inhibit HMG-Co
Reductase, a key enzyme
in cholesterol synthesis
HMG-­‐CoA – statins therefore lower
cholesterol and protect
or against heart disease.
Lovostatin competes with
the substrate for the
active site
lovosta6n HMG-­‐CoA reductase Structural similarity between
the substrate and the inhibitor
Features of enzymes 5. Many drugs inhibit enzymes
Biosynthesis of nucleotides for replicating cells:
Folic acid
Dihydrofolate reductase
nucleotides
enzyme Folic acid
Substrate
methotrexate
structurally similar drug
Features of enzymes 5. Many drugs inhibit enzymes
Biosynthesis of nucleotides for replicating cells:
Folic acid
Dihydrofolate reductase
nucleotides
Toxic to
rapidly
dividing
cells
enzyme methotrexate
Folic acid
Substrate
methotrexate
structurally similar drug
Methotrexate is often used in cancer therapy
Features of enzymes
6. Cellular enzymes can be activated by “phosphorylation”
- the addition of a phosphate group
•  It is other enzymes that actually add the phosphate
group to the other enzyme,
-these enzymes are called kinases
•  Kinases are said to “phosphorylate” the protein
•  Eg: Adrenalin activates Phosphorylase kinase which
phosphorylates Glycogen Phosphorylase.
-This enzyme in turn breaks down glycogen to
release glucose
Signal cascade
Receptor protein Adrenaline (hormone protein) signals Phosphorylase Kinase (protein kinase enzyme) P GP GP Glycogen Phosphorylase Glycogen glucose P =Phosphate group Adrenaline results in the release of glucose which allows for rapid produc6on of energy Understanding Biology
“reductionism”
Reducing down complex reactions to their component parts
genetic engineering
helping biologists understand the finer details of complex functions
“systems biology”
link all of the knowledge that we gain from these techniques together to gain a
greater understanding of how the system as a whole works
“Reductionism”
Biological systems are highly complex ???Can studying each protein separately contribute to our understanding of such complex systems??? ??Would it be possible to chemically synthesise a cell?? It is not enough to just know the components BUT understanding the behaviour of a complex integrated system is incredibly difficult A complex organism can not be analysed without taking it apart Genome sequencing is an example of reduc6onism –  Human Genome Project –  Reduc6onism = reducing complex systems to simpler components that are easier to study –  Human organism breaks down into a code of 3billion leiers “Systems Biology”
Goal: Understand how biological systems are func6onally integrated Systems biology – model of the dynamic behaviour of whole cells High performance computers analyse highly complex systems of known interac6ons “Systems map” of
the interactions
between proteins in
a cell of drosophila.
~3500 proteins
Outer membrane
and cell surface
CELL
Cytoplasm
Nucleus
Eg. of an applica6on: The ability to predict probable side effects of drugs DNA > RNA > PROTEIN > TRAIT
GFP Protein Trait: Green Fluorescence Winner of the nobel prize for chemistry 2008
With the aid of GFP-fusion proteins, we can watch processes that were
previously invisible
GFP as a molecular tool
HIV transmission detected using
GFP-HIV fusion proteins
T cells