Biochem enzymes Flashcards
What are enzymes and what is there function?
Biological catalysts and they speed up the rate at which the point of equilibrium is reached- do NOT change the point of equilibrium
What are enzymes made from?
Mostly protein (ribozymes are made from RNA)
What characterises enzymes?
1) Efficient: work at 37 degrees in solution near neutral pH and increase reaction rate by 10^20
2) Specific: each enzyme has a limited range of substrates and some can distinguish stereoisomers
3) Potent: Each enzyme can convert many substrate molecules to product per second
What is the transition state and how do enzymes effect it?
Transition state is the reaction intermediate species with the greatest free energy.
Enzymes bind and stabilise the transition state
Enzyme substrate complexes are usually unstable. T or F?
True
What are the symptoms of glycogen storage disease?
Hypoglycaemia, liver swelling, skin and mouth ulcers, bacterial /fungal infection
What is glycogen storage disease?
Enzyme deficiency meaning glycogen cannot enter the transition phosphorylated state and cannot be transformed back into glucose. (12 different defects in glucose or glycogen metabolising enzymes)
What are Co-enzymes and co-factors?
Small molecules required by enzymes to catalyse a reaction.
Co-enzymes =organic molecules
Co-factors = inorganic metal ions
What is a apoenzyme and a holoenzyme?
Apoenzyme = enzyme without a co factor Holoenzyme = enzyme with a cofactor
How do cofactors work with enzymes?
Form a metal coordination centre within the enzyme and the enzyme is referred to as a metalloprotein. (Zn, Fe, Cu) Involved in redox reactions
How do coenzymes work with enzymes?
Associate with the enzyme transiently and change charge or structure during the reaction but are regenerated. Some are involved in redox reactions (NAD+ or FAD) others in group transfer processes (CoA or ATP)
What is the name given to tightly bound coenzymes?
Prosthetic groups eg haem in haemoglobin or cytochromes in oxidative phosphorylation
Where are many coenzymes derived from?
Vitamins eg NAD+ and FAD
Why are the symptoms of vitamin deficiencies related to enzyme activity?
Most vitamins function as coenzymes
What is the lock and key model?
Active site of unbound enzyme is complementart to the shape of the substrate
What is the induced fit model?
Binding of the substrate induces a conformational change in the enzyme => complementary fit and formation of enzyme substrate complex
What are isoenzymes?
Isoforms of enzymes that catalyse the same reaction but have different properties and structure eg isoforms of haemoglobin, adult and foetal
When are different isoforms synthesised?
Different stages of foetal and embryonic development
Different isoforms can be present in different tissues and different cellular locations T or F?
True
There are 2 isoforms of lactate dehydrogenase. Where are they found and why is this useful?
1) Heart- premotes aerobic metabolism. Lactate –> pyruvate
2) Muscle- promotes anaerobic metabolism. Pyruvate –> lactate
This is useful as relative amounts can be useful as diagnostic markers
Lactate Dehydrogenase: What can be inferred from lots of M type being produced in the heart?
Must be a hypoxic environment as M subunit is produced in the presence of hypoxic sensitive transcriptional factors
Creatinine Kinase (isoenzyme) is a dimeric protein in the blood which binds to muscle sarcomeres. What type of creatinine kinase is produced normally in the muscle, brain and heart?
Muscle produces M form (MM)
Brain produces B form (BB)
Heart produces M and B to form a heterodimer (MB)
Creatinine Kinase: what does presence of BB in the blood suggest?
Stroke or brain tumour
Creatinine Kinase: what does presence of MB in the blood suggest?
Heart attack
What does a kinase enzyme do?
Add a phosphate group
What does a phosphatase enzyme do?
Remove a phosphate group
How is enzymatic activity regulated reversibly?
Phosphorylation reactions converting the enzyme to the active or inactive form
How is enzyme activity regulated irreversibly?
Irreverable covelent modification => activation of enzymes
or partial proteolysis
What is a zymogen?
Inactive precursor of an enzyme which can be converted to an active enzyme by cleavage of a covelent bond
Trypsinogen and chymotrypsinogen are zymogens produced in the pancreas. Where are they converted to active enzymes?
Small intestine where enteropeptidase cleaves trypsinogen to form acitve trypsin which cleaves chymotrypsinogen to chymotrypsin
How does the substrate concentration [S] effect the rate of the catalysed reaction V.
What is V?
What is Vmax?
What is Km?
V = moles of substrate converted to product per second
Vmax is where there is no limiting substrate
Km is the substrate concentration when the rate is 50% of Vmax.
What is the michaelis-menton constant and what does it explain?
PLEASE SEE SHEET
Km= (K-1 +K2) / K1
The relationship between Vmax and Km
The michaelis-menton model.
What is K1?
What is K-1?
What is K2?
K1= forwards RATE CONSTANT for enzyme association with the substrate K-1= the backwards rate constant for enzyme dissociation with the substrate K2= forwards rate constant for enzyme conversion of a substrate to product
How can you measure Vmax and Km?
NB: not accurate as the velocity will never reach Vmax.
1) Measure the initial rate of reaction at a known substrate concentration
2) Repeat at increasing substrate concentrations
3) Plot initial reaction rates as functions of substrate concentration
4) At infinite substrate concentration the initial rate approaches V max
5) Km is the [S] at 50% of V max
What is the michaelis menton equation?
V = Vmax[S] / (Km + [S]). If you take the recepricle you get a straight line and accurately determine Vmax and Km.
Describes the rate of catalysis as a function of substrate concentration.
On a lineweaver-burk plot, where is Vmax and Km found?
Vmax = intersection with Y axis Km = intersection with the X axis
What is implied by a low and high Km?
Low Km = enzyme only need a little substrate to work at half maximal velocity (efficient)
High Km = enzyme needs lots of substrate to work at half maximal velocity (less efficient but more sensitive and responsive)
What is MODY (maturity onset diabetes of the young)?
Loss of glucokinase activity => loss of insulin mediated glucose homeostasis.
Hexokinase (RBC) and Glucokinase (Liver and Pancreas) both catalyse the same reaction: Glucose + ATP –> ADP + Glucose-6-phosphate. Which has a lower Km and why?
Hexokinase has a low Km so energy production is maintained even if glucose levels fall
Glucokinase has a high Km meaning if glucose levels fall less glucose will be used by the liver and converted into glycogen (enables glucose sensing). Glucose abundance in liver is regulated by insulin and only excess glucose metabolised
Proline hydroxilases regulate hypoxia inducable transcriptional factors. Does it have a high or low Km for oxygen?
High Km which allows it to be sensitive to levels of hypoxia over physiological ranges of oxygen
What is orthosteric inhibition?
Inhibition at the same site eg competitive
What is allosteric inhibition?
Inhibition at a site other than the active site inducing a conformational change
Can irreversible inhibition be competitive?
No
What happens to Vmax and Km in competitive inhibition?
Vmax = same Km = varies
Explain methanol poisoning by competitive inhibition?
1) Methanol is the substrate for alcohol dehydrogenase
2) Causes severe tissue damage and blindness by conversion to formaldehyde and drives metabolic acidosis
3) Alcohol dehydrogenase Km for ethanol is 20x greater than for methanol
4) Treat patients with 40% ethanol, dialysis and ventilation
What happens to Vmax and Km in non competitive inhibition?
Vmax = lower Km = same
Do allosteric enzymes follow Michaelis menton kinetics?
No- increasing the substrate concentration results in a sigmoid curve eg binding of oxygen to haemoglobin
What is the most common mechanism of feedback inhibition?
Allosteric control using end products to inhibit the upstream rate
