Enzyme Kinetics Flashcards
What is V max?
The maximum velocity of a reaction (when all the enzyme has been used)
What is Km?
The concentration of substrate which is where Vmax is at 50%
In the reaction E + S = ES what are the two rate constants?
Forwards is K1 (enzyme association) and backwards is K-1 (enzyme dissociation)
In the reaction ES = E + P what represents the forward equation?
K2 - rate constant of enzyme conversion of substrate to product
What is the Michaelis Constant (Km)?
Km = (K-1 + K2)/K1
What is the Michaelis-Menten model?
[ES] = K1[E][S]/(K-1+K2)
How are Vmax and Km measured?
Measure initial reaction velocity (V0) at a known substrate concentration and repeat at increasing substrate concentration
How do you plot Vmax and Km on a graph?
Initial reaction rates (V0) are plotted as a function of substrate concentration. At infinite substrate concentration the initial reaction rate approaches a maximal rate Vmax. Take half of Vmax = Km
What are Vmax and Km as straight line components?
Vmax is the Y axis interception and Km is the x axis interception
How does level of Km affect substrate concentration?
Low Km means only a little substrate required and a high Km means a lot of substrate for half-maximal velocity
How do red blood cells and liver&pancreas both catalyse glucose conversion with different Km?
RBC: Hexokinase Km is 0.05mM glucose which maintains energy production by glycolysis even if glucose levels drastically fall
L&P: Glucokinase Km is 5mM glucose which enables glucose sensing and homeostasis. It’s abundance in the liver is regulated by insulin and excess blood glucose is metabolised
What is MODY?
Maturity Onset Diabetes of the Young - loss of glucokinase activity = loss of insulin-mediated glucose homeostasis
What is the transcription factor in oxygen regulation pf gene expression?
Hypoxia inducible factor
What is the mechanism in oxygen regulation of gene expression?
Oxygen sensors (prolyl hydroxylases) –> transcription factor (hypoxia inducible factor) –> genes for surviving hypoxia (RBC synthesis, blood vessel growth and anaerobic survival pathways)
Do proline hydroxylases have a high or low Km?
High which enables oxygen sensitive oxygen sensing over physiological ranges of PO2
What are the problems with Monge’s disease?
Excessively high haemocrit, loss of arterial O2 saturation and loss of O2 regulation of Epo production
What is Von Hippel Lindau Syndrome?
Loss of propyl hydroxyls activity which causes excessive blood vessel growth (angiogenesis) leading to haemangiomas in the brain and subcutaneous haemangiomas in the track spinal chord
What is competitive inhibition?
A reversible inhibition in which the inhibitor binds to the active (catalytic) site and blocks substrate access - Orthosteric
What is reversible non-competitive inhibition?
The inhibitor binds to a site other than the catalytic centre causing a conformational change which stops the enzyme from binding - Allosteric
What is irreversible non-competitive inhibition?
Usually involves the formation or breakage of covalent bonds in the enzyme complex
What affect does competitive inhibition have on Vmax and Km?
Vmax doesn’t change but Km varies
What is the substrate for alcohol dehydrogenase (ADH)?
Methanol
What does methanol poisoning cause?
Severe tissue damage and blindness by conversion to formaldehyde. (also drives metabolic acidosis)
Is the ADH Km higher for ethanol or methanol?
Ethanol (by 20x)
How do you treat methanol poisoning?
Treat patient with 40% ethanol in combination with dialysis and ventilation
How does non-competitive inhibition affect Km and Vmax?
Vmas varies but Km does not change
What is feedback inhibition?
Inhibition of rate limiting enzymes by end products (common form of allosteric control)
Why is feedback inhibition important?
Avoids the build up of intermediates in the pathway which can be highly reactive and therefore biochemically dangerous
Increasing substrate concentration with allosteric enzyme produces what kind of curve?
Sigmoidal (not hyperbola like with Michaelis-Menten)
What is allosteric enzyme’s co-operative behaviour?
Allosteric factors modulate enzyme kinetic behaviour
What can control allosteric enzymes?
Allosteric inhibitors and allosteric activators
What is a major example of allosteric regulation?
The binding of oxygen to haemoglobin as it shows positive co-operativity and has multiple controllers (protons, CO2, 2,3 biphosphateglycerate)
