Enzyme regulation Flashcards
Explain in short how enzymes work.
By stabilizing the transition state, and thereby lowering the activation energy for the reaction. This is done by creating a microenvironment in the active site, with specific residues sticking out that matches the transition state. This can be by excluding water to maximize non-covalent interactions or by having many weak non-covalent interactions that in unison stabilize the transition state. This functions as an “entropy trap” that is paid for by the many weak interactions that lowers the activation energy.
Enzymes are tightly regulated, why?
The concentration of things in a living cells are far from equilibrium, and as enzymes lower the rate of reactions by many orders, it’s important to regulate them to keep the levels of everything where the cells need it to be. It’s more energetically economic to only catalyze reactions when the products are needed.
One type of enzyme regulation (sort of indirect) is substrate level control. Explain what it is.
Enzymes with first order kinetics (rate only dependent on the substrate concentration) is under substrate level control. This means, that the more substrate, the faster the reaction and vice versa.
This is utilized in muscle tissue for example, where the enzyme reaches max velocity fast to be able to act fast, while a homologous enzyme in the liver has more levels of regulation, it needs to respond differently to different substrate levels.
There are four types of direct enzyme regulation, which? Explain them in short.
- Allosteric regulation: the reversible, noncovalent binding of regulatory compounds called allosteric effectors (activators or inhibitors) to allosteric sites, which causes a conformational change in the enzyme, converting it to a more-active (activator) or less-active (inhibitor) form. A structural transition between R (relaxed) active form and T (tense) inactive form.
- Covalent modification: The transfer of one or more functional group to the specific residues of the enzyme, causing it to be activated/inactivated. When an amino acid residue in an enzyme is modified, a novel amino acid with altered properties has effectively been introduced into the enzyme. The most common is phosphorylation, but could also be adenylation, methylation or other modification.
- Proteolytic cleavage: The irreversible severing of a part of the enzyme, activating/inactivating it.
- Some enzymes are stimulated or inhibited when they are bound by separate regulatory proteins.
There are two types of allosteric regulation, which?
Homoallostery: The substrate or product of the enzymatic reaction as effector
Heteroallostery: another molecule acts as effector, for example, ammonia accumulation is a sign of starvation (produced when amino acids are broken down) and could activate a pathway that creates a better energy form.
If you compare allosteric regulation and covalent modification in terms of effect and responsiveness to changing conditions, how do they compare?
Allosteric regulation give smooth and highly responsive regulation possibilities, as the concentration of the effector have a direct effect. Lower concentrations of the effector, less allosteric regulation and vice versa. It’s kind of like a dimmer.
Covalent modification on the other hand is sort of an “on/off switch”. Where another enzyme turns another on and off depending on other signals.
Allosteric regulation is more or less a rule for enzymes catalyzing energetically expensive reactions.
Give an example of an allosteric effector and what it does to the enzyme.
CTP is an allosteric inhibitor of aspartate transcarbamoylase (which is part of the nucleotide synthesis pathway). When bound to the regulatory subunits, CTP stabilizes the T (less active) state which hides the active sites. When released, the enzyme converts to the R (more-active) state and the active sites are made available for substrate to bind.
How can you experimentally identify allostery?
look up!
For enzymes with allosteric regulation, their kinetics doesn’t follow Michaelis-Menten kinetics and instead form a sigmoid curve. What parameter is used instead of Km?
K 0,5 = concentration at half saturation. The T-state have a much higher K 0,5 and the S-state much lower, and in between there’s a gradient.
Which is the most common form of covalent modification? How does it work in detail?
Over 500 different types of covalent modification have been identified in proteins, but the most common one is phosphorylation.
In phosphorylation a phosphoryl group (PO3, with one double bound O and two single bonded O-) is transferred from a donor (usually ATP) to the hydroxide of Tyr, Ser, Thr or His in a reaction catalysed by a kinase. The reverse reaction is catalyzed by a phosphatase. The addition of the phosphoryl group results in a big structural change, which can activate or inactivate an enzyme. Many enzymes needs several phosphorylations to be activated and some enzymes are phosphorylated by many different kinases, eg glycogen synthase.
Provide an example of covalent modification.
Phosphorylase B is the inactive form of an enzyme that cleaves off individual glucose monomers from glycogen. It can be activated by phosphorylation catalyzed by phosphorylase kinase (with 2ATPs as phosphoryl donor). The double phosphorylation converts the enzyme to it’s active form; phosphorylase A. Since phosphorylation is a reversible process, the enzyme can be deactivated by phosphorylase phosphatase which utilize H2O as an oxygen donor to dephosphorylate the residues and yield 2Pi.
Name one other common covalent modification and explain it in short.
Adenylylation is the addition of an adenosine monophosphate to a residue (Tyr), a major structural change!
Explain how proteolytic cleavage works with an example.
Proteolytic cleavage is a form of irreversible regulation, mostly utilized in the breakdown of food which needs potent enzymes. The problem is that if a cell synthesize an enzyme capable of breaking down protein for example, what’s preventing that enzyme to break down the cell itself? The solution is to synthesize the enzyme in an inactive form (zymogen).
An example of this is Trypsin, which is synthesized as trypsinogen in the cell, transported out and then an enteropeptidase cleaves away a part of the peptide chain, which converts it to its active form, trypsin!
Explain the term “substrate cycling”.
Substrate cycling is a way of ensuring that irreversible reactions can always be regulated. Say you have a molecule R, which allosterically regulates a kinase that converts R to molecule P (product). If R runs out, then there is no way to regulate the kinase, so there is another reaction that uses ATP to convert P to R, so that regulation can occur if needed.
Provide an example of substrate cycling.
A good example of substrate cycling is phosphofructokinase-1 (PFK-1) which is a key enzyme in glycolysis, catalyzing the reaction from F6P to F1,6-BP (irreversible). Say that we need to run the reaction, but we don’t have any substrate, then there’s another reaction where FBPase-1 hydrolyses F1,6BP to F6P + P which produces the substrate so we can run the reaction again.
In the sum of these two reactions, ATP + H2O → ADP + Pi („futile“ cycle) we only use ATP and get “nothing” out of it net, but in practice it’s used to increase the flux through irreversible reactions which leads to increased sensitivity to effectors.
