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58 Cards in this Set
- Front
- Back
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Two things which can cause enzymes to malfunction are...
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Genetic defects and poisons/toxins
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What is a kinetically favourable reaction?
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One which releases free energy.
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How do enzymes allow reactions to occur at a greater rate?
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By overcoming the (kinetic) energy barrier to be overcome.
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State four extremes which may be required for a reaction in the absence of an appropriate enzyme.
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pH, temperature, pressure and concentration.
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What do enzymes do to the circumstances required for a reaction to take place?
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Decrease required temperature and concentration, and move required pH to nearer physiological pH (blood has pH of 7.4 - slightly alkaline).
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Proteolytic enzymes increase the rate of reactions in the gut by ... times.
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10^5
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Urease increases the rate of urea breakdown by ... times.
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10^14
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Enzymes are proteins with two important characteristics. These are:
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1. They catalyse biochemical reactions - but remain unchanged at the end of the reaction.
2. They control these reactions in terms of specificity and rate. |
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Mass action equation
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K = [B]/[A]
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Enzymes can promote a reaction in 3 ways. These are:
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Covalent catalysis, acid/base catalysis and orientation.
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Give an example of an enzyme which uses orientation to overcome the energy barrier
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Lactate dehydrogenase
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Give an example of a group of enzymes which use both acid/base catalysis and covalent catalysis to overcome the energy barrier.
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Serine proteases
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What two things are enzymes very specific for?
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The structure of their substrates and the types of reactions they catalyse.
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Name an enzyme, and provide chemical formulae to back this up, which has a very high level of specificity.
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Urease:
Hydrolyses urea (NH2-CO-NH2) but not CH3-NH-CO-NH2 |
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Name an enzyme, and provide chemical formulae to back this up, which has a more general level of specificity.
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Proteolytic enzymes such as trypsin (hydrolyses peptide bonds where R1 is arginine or lysine - positive), chymotripsin (likes bulky groups on R1) or pepsin (R1 may be Asp or Glu, R2 may be Tyr or Phe).
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What is rate of reaction determined by?
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Time course of the reaction, substrate concentration, enzyme concentration, pH, temperature.
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At what temperatures are enzyme assays usually carried out at?
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25 degrees C or 37 degrees C
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Name an enyme which can stand extremes of temperature
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Phospholipase A2
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What is a co-enzyme?
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A molecule possessing physiochemical properties not found in the polypeptide chain of the enzyme that acts together with the enzyme to catalyse a chemical reaction.
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What are the two types of co-factors?
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Co-enzymes and metal ions.
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Name three examples of co-enzymes.
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ATP, NAD+, NADP+
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What is a holoenzyme?
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A metalloenyme with its metal ion component.
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What is an apoenzyme?
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A metalloezyme without its metal ion component.
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What type of reactions are metal ions involved in?
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Oxidative and hydrolytic reactions.
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Enzyme activity is expressed in 2 units- 'Units' and 'Katals'. What do these represent?
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1 unit = 1 mol per minute
1 Katal = 1 mol per second (6 x 10^7 units) |
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What are the advantages of commercial enzyme assay kits?
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Reliable, reproducible, widely available - can be accurately reproduced in many labs in different places.
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What are the basic requirements for standard conditions?
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Optimum pH, 25 degrees C, saturating substrate.
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In order to be suitable for use in diagnostic assaying, an enzyme should have 2 important characteristics. These are...
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... to be easy to assay, and specific to a single/very small range of tissues, ideally a single one.
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Where are high levels of alkaline phosphatase enzyme found?
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Biliary canaliculi of the liver.
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Why is alkaline phosphatase easy to assay?
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it has a very wide specificity - that is to say it can function relatively easily with a wide range of substrates, This makes it easy to assay.
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What indication of rate do we measure when assaying alkaline phophatase?
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Colour change is measured, using a spectrophotometer.
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Why is alkaline phosphatase assayed at pH 10.5?
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To ensure the product is unprotonated, and therefore coloured yellow, allowing colour change to be monitored throughout the reaction.
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Why is assaying for alkaline phosphatase in the blood an unreliable indicator of liver damage in young animals?
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Alkaline phosphatase is also found in bone. As you animals have high osteoblastic activity, where bone is broken down and formed at a great rate, levels of alkaline phosphatase in the blood will be high, even in the absence of liver damage.
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Where are the enzymes alanine transferase (ALT) and aspartate transaminase (AST) found in equal concentrations in the body?
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The liver.
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How is the concentration of ALT in a blood sample tested, as the substrate/product is not coloured?
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Alanine + alpha-oxoglutarate (in the presence of alanine transferase) gives pyruvate + glutamate. Pyruvate is then reacted with NADH and H, in the presence of lactate dehydrogenase, to give lactate and NAD+. NAD+ and NADH absorb light differently at 340nm, therefore allowing the progress reaction to be monitored.
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Why are kits for the assay of aminotransferases such as ALT so expensive?
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They require a complicated reaction mixture, including the enzyme lactate dehydrogenase.
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What can assaying of alanine transferase (via the use of lactate dehydrogenase) be used to diagnose?
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Canine hepatitis, liver cancer and carbon tetrachloride poisoning.
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Why is ALT not assayed in large animals to diagnose liver damage?
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Large animals contain little hepatic ALT - even with extensive liver damage, not a lot will be released.
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As LDH is found in many different body tissues, is it possible to determine where in the body it is from? If so, how?
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LDH occurs in different forms in different parts of the body. Native electrophoresis is used to analyse these, and therefore determine which tissue is damaged.
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How many parts is the enzyme LDH made up of?
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It is a tetramer (composed of four protein monomers bound together). The monomers occur as a number of different isozymes (isoenzymes).
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What are the most common monomers of LDH? Where are they found?
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M - found in skeletal muscle - and H - found in cardiac muscle.
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As there are two monomers, and LDH is made of four parts, how many potential forms of the enzyme are there?
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5 - H4, H3M, H2M2, HM3, M4
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Which LDH variations (H4, H3M, H2M2, HM3, M4) would you expect to find in the blood if there was damage to the heart?
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H4 primarily, with the gradient dropping off to almost no M4 present.
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Which LDH variations (H4, H3M, H2M2, HM3, M4) would you expect to find in the blood if there was damage to the adrenal glands?
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Primarily H2M2 - gradient dropping off to almost no H4 or M4.
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Which LDH variations (H4, H3M, H2M2, HM3, M4) would you expect to find in the blood if there was damage to skeletal muscle?
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Primarily M4, gradient dropping off to almost no H4.
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Which LDH variations (H4, H3M, H2M2, HM3, M4) would you expect to find in blood serum?
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H4 primarily, with the gradient dropping off to almost no M4 present.
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Where is creatine kinase found?
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Muscle tissue and the brain.
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What reaction does creatine kinase catalyse?
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The exchange of phosphate from one store to another, e.g. creatine-Pi + ADP to (and from) creatine + ATP. This is a very easy way to regenerate ATP.
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Why do we not use enzyme assays to diagnose brain damage?
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Because the blood-brain barrier prevents enzymes from brain tissue entering the blood stream, except in cases of catastrophic damage, in which case assaying would most likely not be necessary as the damage would be obvious.
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What might we use assaying of LDH to diagnose?
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Myocardial infarction in humans, rhabdomyolysis in horses and greyhounds and hereditary muscular dystrophy in humans.
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What is rhabdomyolysis?
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When animals are trained and exercised to a high level of performance, the vasculation of their muscles is increased. When these animals are on decreased exercise, such as lame or on box rest, the level of vasculation decreases. If the animal is restarted too rapidly, the muscles will require more oxygenated blood than can reach them, resulting in a painful and damaging build-up of lactic acid in the tissue.
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As the creatine kinase reaction is not coloured, which other two reactions are required?
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The reaction of glucose with ATP to give glucose-6-P and ADP, and then the reaction of glucose-6-P and NADP+ to give 6-phosphogluconolactone, NADPH and H+.
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Write out the equations for the three linked reactions used to assay creatine kinase.
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Creatine-Pi + ADP <=> Creatine + ATP
Glucose + ATP <=> Glucose-6-P + ADP Glucose-6-P + NADP+ <=> 6-phosphogluconolactone + NADPH + H+ |
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How do we therefore use the three linked reactions to monitor the level of creatine kinase activity?
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NADPH and NADP+ have different absorptions at 340nm - monitoring the activity of these allows us to monitor the levels of creatine kinase activity.
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What is n-acetyl cisteine used for in assaying CK?
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Two sulphydryls in the active site of CK oxidise to form disulphide bridges in the oxidising conditions of the bloodstream. This inactivates the enzyme. Conditions in the cell are reducing enough to avoid it. N-acetyl cisteine is a reducing agent, and prevents this from happening.
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Why is Vi in this reaction 0?
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There is a time delay - it occurs after a couple of seconds.
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Under what circumstances might you see a normal increase of CK, up to about 5x? What effect can this have on blood sampling?
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After excercise - therefore samples must be collected no earlier than one hour after exercise.
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How is it possible to track the development of rhabdomyolysis?
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Both LDH and CK are used in combination. CK peaks at about 1 day, whereas LDH peaks later, at about 4 days. If both after sampled for over a period of a few day it is possible to track the progress of the pathology.
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