Enzymes Flashcards
What does a co-factor do?
Binds to the enzyme to contribute to the catalytic mechanism in some way
What does an enzyme do?
Catalyses the rate at which a chemical reaction reaches equilibrium, without changing the position of equilibrium
What is Kcat?
The number of substrate molecules a single molecule of enzyme can bind and convert every second (when substrate is unlimited)
What three things can be measured in an enzyme assay to measure enzyme activity?
Rate of production of product
Rate of loss of substrate
Rate of production or loss of cofactor
Why is it important to meaasure the initial rate?
Rate can decrease due to less substrate being available as the reaction goes on
How does gel filtration work?
There is a column of beads with small cavities/pores. Smaller molecules can move through/get stuck in these pores and so take a long time to fall through the column. Larger molecules fall straight through. This layers the molecules by size
What is specific activity?
The number of enzyme units per mg of protein
What two things do we want to maximise in the purification of an enzyme?
Recovery of enzyme
Purity of enzyme
How do you calculate specific activity?
crude (or purified) rate in um per min / protein per ml in the sample
How do you find % recovery of an enzyme?
units in pure sample / units in crude sample x 100
How do you find the degree of purification of an enzyme?
specific activity of pure sample / specific activity of crude sample
What are the properties of an irreversible enzyme inhibitor? 3
Covalent or tight binding
Can be toxins that kill enzymes
Can be therapeutic (eg aspirin inactivating COX)
What are the properties of a reversible enzyme inhibitor? 4
Competitive
Can be overcome at high [S]
Vmax unaffected
Km increased
What are the properties of a non-competitive inhibitor? 5
Binds to a site other than the active site (allosteric)
Substrate and inhibitor can bind at the same time
Active site shape can be altered
Enzyme activity reduced
Vmax decreased, Km unaffected
What is the equation for a Lineweaver-Burk plot?
1/V = KM/Vmax x 1/[S] + 1/Vmax y = m x X + c
What do the lines on a linear plot look like in competitive enzyme inhibition?
The line with the inhibitor has a higher Km than the line without. Both lines have the same intercept (Vmax)
What do the lines on a linear plot look like in non-competitive enzyme inhibition?
Both lines have the same Km (starting point). The inhibited line has a lower Vmax (higher intercept).
What happens during the transition state of a reaction? 3 What do catalysts do to this state? 1
Higher free energy than substrate or product
Unstable
Slows down the reaction
A catalyst can lower the transition state energy and stabilise it
How can RNA be hydrolysed by acids or bases?
Contains O and OH
Both cleave phosphate ester bonds
Acid O -> OH
Base OH -> O-
How do RNA histidine side chains allow for cleavage by both base and acid at the same time? When is each more likely to happen?
Histidine can be protonated or unprotonated around its pKa.
Hydrophobic environment = unprotonated
Negative environment = protonated
What is electrostatic steering?
Some enzymes can attract the substrate towards the active site. E.g using positively charged groups
What does chymotrypsin do?
Cleaves peptide bonds on the C terminal side of bulky hydrophobic + aromatic amino acids
What does trypsin do?
Cleaves peptide bonds on the C terminal side of K or R amino acids
What does elastase do?
Cleaves peptide bonds on the c terminal side of small amino acids
Name 4 uses of serine proteases
Control of blood clotting
Immune response
Penetration of ovum
Protein turnover regulation
What are the basic principles underlying burst kinetics?
Enzyme reactions can produce an ES complex as well as multiple products. If two (or more) products are produced, the one that does not form the ESC will be released quickly causing a ‘burst’. It will then take more time for the second product to dissociate from the ESC in the steady state phase.
What is the importance of a specificity pocket?
A space near the active site that enables the enzyme to be substrate specific?
What are three examples of specificity pockets?
Chymotrypsin pocket (large and hydrophobic) Trypsin (Asp 189- only allows positively charged K + R) Elastase (bulky Val molecules only allow small amino acids)
How does the body stop serine proteases from breaking important peptide bonds?
Inactive serine protease precursors called zymogens are packaged into vesicles and released when required. These can be activated by the cleavage of a specific peptide bond.
Which enzyme cleaves the peptide bond required to activate serine protease precursors?
Trypsin
What activated inactive trypsin (trypsinogen)?
Enteropeptidease secreted by the cells lining the duodenum.
What is the importance of having one anzyme control the activation of all serine proteases?
Means that the whole process can be switched on and off.
On by enteropeptidase and off by pancreatic trypsin inhibitor.
How have serine proteases come about by divergent evolution?
An ancient single gene containing the catalytic triad, over time, duplicated and evolved to form different mutations and features.
What is K?
The rate constant
What does K[A] find?
Rate
What is K1 and K2?
The rate of the forwards vs reverse reaction
What is V?
Velocity
At equilibrium V=?
0
At equilibrium K1[A]=?
K2[B]
At equilibrium [B]/[A] = ?
K1/K2 OR Keq
What is V=Vmas[S] / [S] + Km?
The Michaelis-Menten equation
How does HIV protease inhibit enzymes?
Converts viral proteins into acitve forms by cleaving specific peptide bonds.
Important in the life cycle of HIV
What does Crixivan do to HIV?
- similar structure to the peptide substrates
- binds to HIV protease reversibly
- inhibits HIV protease
What are the properties of irreversible inhibitors?
- covalent or tight bindig
In a Lineweaver–Burk plot, the presence of a competitive inhibitor will alter: (3)
The slope of the curve
the intercept on 1/s
What can reduce the effect of a competitive inhibitor of an enzyme?
Increased substrate concentration
