Regulation of Protein Function Flashcards
What is the difference between the active site and an allosteric site?
ACTIVE SITE = specific region of enzyme where a substrate/ligand binds and catalysis takes place
ALLOSTERIC SITE = specific region in a multi-subunit enzyme where a substrate/ligand binds, influencing the subsequent binding of ligands/substrates at another subunit
What is the difference between a holoenzyme, apoenzyme, zymogen, and isozyme?
HOLOENZYME = active enzyme + non-protein component (coenzyme)
APOENZYME = inactive enzyme ( - non-protein component)
ZYMOGEN = inactive enzyme precursor (proenzyme)
ISOZYME = enzyme catalysing the same reaction but differing in amino acid sequence (and therefore in reaction kinetics & affinities for substrates)
How do enzymes catalyse reactions?
Reaction can proceed along an alternate pathway with a lower activation energy than the uncatalysed reaction.
The transition state is stabilised.
What is the activation energy (Ea) of a reaction?
Amount of energy (J) needed to convert all the molecules in 1mol of a reactant from a ground state to the transition state
i.e. energy needed for reaction to progress
(kinetic energy when substrate molecules collide in the correct orientation)
What factors can affect the reaction velocity?
- [Substrate]
- Temperature:
+ = increase in molecules with sufficient kinetic energy to reach Ea (increase in rate)
+++ = denaturation of enzymes (decrease in rate) - pH = affects ionisation of particular chemical groups which may be involved in the reaction (increase or decrease rate; until denaturation)
Define Vmax, Km, the activity of an enzyme, and enzyme units.
Vmax = maximum velocity (all enzyme active sites are saturated with enzymes)
Km = substrate concentration at 1/2Vmax
Activity of an enzyme = amount of substrate converted to product over time (mol/min)
Enzyme unit = amount of enzyme (umol) that converts 1mol of product per minute under standard conditions (per litre of serum or per gram of tissue)
Note: multiplying standard rate leads to the same value
Give some examples of irreversible and reversible inhibition.
Irreversible = molecule binds tightly (covalently) and cannot be easily removed e.g. nerve gas (sarin), cyanide
Reversible = molecule binds loosely (non-covalently) and can freely dissociate
What are the effects of competitive and non-competitive inhibition on Vmax and Km? Give some examples of drugs for each one.
COMPETITIVE INHIBITION = inhibitor resembles substrate and is partially complementary to the active site (reduces [ES] )
Vmax = SAME (as long as [S] is high enough)
Km = INCREASES (more substrate needed to compete with inhibitor for active sites)
e.g. Statins
NON-COMPETITIVE INHIBITION = inhibitor binds at allosteric site (therefore multi-subunit enzyme) and prevents substrate from binding at active site (reduces enzyme turnover)
Vmax = DECREASES (fewer enzyme molecules can react)
Km = SAME (substrate conc. at 1/2Vmax is the same)
e.g. ACE inhibitors
Give some examples of short-term and long-term protein regulation.
SHORT-TERM: Change substrate/protein concentration Change enzyme concentration Allosteric regulation Covalent modification Proteolytic cleavage
LONG-TERM:
Change in rate of protein synthesis (e.g. gene transcription level)
Change in rate of protein degradation (ubiquitination)
What is the difference in the kinetics and location of glucokinase and hexokinase?
GLUCOKINASE:
liver & pancreas
High Vmax, high Km (low affinity for glucose)
Sigmoidal
HEXOKINASE:
most tissues
Low Vmax, low Km (high affinity for glucose)
Hyperbolic
How does allosteric regulation work?
Multi-subunit regulation only
Sigmoidal regulation
Activators: increase proportion of enzyme in R state
Inhibitors: increase proportion of enzyme in T state
What are some examples of covalent modification? What are the donor molecules, modified proteins, and protein functions in these examples?
PHOSPHORYLATION:
Donor molecule = ATP
Modified protein example = glycogen phosphorylase
Protein function = glucose homeostasis, energy transduction
ACETYLATION:
Donor molecule = Acetyl CoA
Modified protein example = histones
Protein function = DNA packing, transcription
MYRISTOYLATION:
Donor molecule = Myristoyl CoA
Modified protein example = Src
Protein function = signal transduction (membrane proteins)
What is the difference between reciprocal regulation and feedback inhibition?
RECIPROCAL REGULATION = prevents concurrent activity in two closely parallel pathways
FEEDBACK INHIBITION = end product of a reaction inhibits an allosteric enzyme involved previously in the reaction
What are some examples of enzymes activated by specific proteolytic cleavage?
Activation of zymogens e.g. pepsinogen —–> pepsin
Prohormones e.g. proinsulin ——-> insulin
Blood clotting cascade
Tissue remodelling processes
Apoptosis mediated by caspases which are synthesised as procaspases.
How is protease activity regulated?
Endogenous inhibitors e.g. alpha-1-trypsin
Protein degradation: ubiquitin-protease pathway
