Enzymes I Flashcards
What are enzymes?
Proteins that speed up (catalyse) specific chemical reactions
List 3 functional areas of enzymes.
Digestion- carbohydrates, fats, proteins
Blood clotting- fibrin clot catalysed by thrombin
Defence-immune system- activation of complement
Movement- muscle actomyosin is an ATPase
Nerve conduction- membrane pumps for Na+, K+, Ca2+
What are some enzyme defects that cause diseases?
These involve an amino acid, a sugar and a complex lipid
Phenylketonuria- cannot convert Phe to Tyr
Glycogen storage- cannot mobilise glucose
Tay-Sachs disease- defect in processing a membrane ganglioside leading to neuronal damage and death
What are some different types of enzymes and what do they catalyse?
Proteases hydrolyse peptide bonds so they have a protein as their substrate
Nucleases break down nucleic acids (RNA and DNA)
Polymerases take simple building blocks and link them together to make polymers
Kinases transfer a phosphate from PTP onto a substrate
How are enzymes drug targets?
Antibiotics- e.g. penicillins inhibit cell wall synthesis
Anti-inflammatory agents- aspirin blocks prostaglandin
Anti-cancer drugs- methotrexate is a folate analogue; interferes with the synthesis of DNA precursors
What are some key enzyme properties?
Increases reaction rate by up to 10 billion fold
Show specificity
Unchanged at the end of the reaction
Do not alter reaction equilibrium
Facilitate reaction by decreasing the frequency activation of the reaction
Enzymes speed up reactions by reducing ΔG‡- free energy of activation. But how?
The active site is a 3D cavity or cleft that binds substrate(s) with specificity through electrostatic, hydrophobic, hydrogen bonding and Van der Waal’s interactions
Formation of an enzyme-substrate (ES) complex at the active site is the first step in enzyme catalysis
What is the evidence for active sites?
X-ray crystallography (usually done at synchrotrons):
- make a large amount of the protein by recombinant DNA tech means
- Get it to crystalise
- Once you have crystals put them into an intense beam of X rays (at a single wavelength) (the wavelength of X rays is similar to the chemical bond length)
- And so when you shine X-rays and if you mount a detector or a film on the other side of the crystal you get a diffraction pattern
- If you can store and process the pattern of spots you can work out the structure of the molecules in the crystal
What factors are responsible for enzyme catalysis?
Enzymes reduce ΔG‡ and speed up the reaction by using the enzyme-substrate binding enzyme:
To bring molecules together in the active site A+B= C+D
To constrain substrate movement
To strain particular bonds in the substrate making breakage easier. Substrate is distorted on binding to resemble the transition state
To stabilise positive and negative charges in the transition state
TO exclude water from the active site- makes reaction go faster
To provide a reaction pathway of lower energy e.g. involving covalent enzyme-substrate intermediates
Use cofactors- bring new chemistry to the active site with NAD(H), FAD(H2), metal ions such as Mg2+
Look up and draw the enzyme kinetics equation and graph.
(In addition to the equation and actual graph) Y-intercept= 1/Vmax Slope= Km/Vmax X-intercept= -1/Km Y axis= 1/V X axis= 1/[S]
How are Vmax and Km useful kinetic parameters?
Vmax and Km are useful kinetic parameters
Vmax/[enz] i.e kcat* Km
Lysozyme 0.5 s-1 6 x 10-6 M
Carbonic anhydrase 800,000 s-1 8 x 10-3 M
*Turnover number – max no of substrate molecules handled per active
site per second. Carbonic anhydrase is a very efficient enzyme. The
average kcat for most enzymes is ~10 s -1.
Values for Km are usually in the µM to mM range. Km is a measure of
the affinity of the substrate for the active site
What happens during competitive inhibition?
Inhibitor I competes with substrate S for binding to the enzyme active site forming an inactive EI complex
In the presence of a competitive inhibitor: Km is increased (it takes more substrate to achieve Vmax/2)
However, Vmax is unaltered as the effects of the inhibitor can be competed out at high substrate concentrations
What happens during non-competitive inhibition?
Inhibitor I binds at a different and does not compete with substrate S for binding at the active site.
Therefore, in the presence of a non-competitive inhibitor, the substrate Km is unaltered; but the Vmax is reduced
How is enzyme activity regulated?
Control of gene expression -enzyme amount
Compartmentation: sequences in enzyme polypeptide chain target enzyme polypeptide chain target enzyme to ER, mitochondrion, nucleus etc
Allosteric regulation:
A regulatory molecules (acting at a pocket distinct from the active site) changes the enzyme conformation to influence the active site and decrease (or in some cases increase) enzyme activity
Controls the flux of material through a metabolic pathway
Covalent modification of enzyme- enzyme changes shape and activity e.g. phosphorylation
How does feedback inhibition regulate metabolic pathways?
Control achieved by allosteric regulation-glycolysis has multiple control points including the early enzyme phosphofructokinase which is regulated by citrate, ADP and ATP
