5: Enzymes Flashcards
what are enzymes
biological catalysts that speed up the rate of a reaction without itself being changed in the process
specific for the substrates based on their structure
most proteins are enzymes
important for metabolism, movement, digestion, gene expression
increase reaction rates by 10^17
disease
malfunctions in enzymes disrupt homeostasis
under or overproduction of a single enzyme mutates DNA resulting in disease
a single amino acid substitution can destabilise structure and disrupt binding sites
cancers come from mutations in enzymes that regulate cell cycle control or DNA repair mechanisms
enzyme structure
mainly globular proteins (sphere shaped)
amino acid sequence specifies 3D shape
work at optimum ranges of pH and temp otherwise they denature
active site includes the binding site (binds and orients the substrate) and catalytic site (reduces chemical activation energy)
active site
highly specific for their substrate
form a cleft/crevasse on surface of enzyme
binding: lock and key model
geometric fit
considered rigid and fixed
substrate fits into enzyme like a key into lock
binding: induced fit model
substrate induces a conformational change on binding
dynamic interaction between enzyme and substrate
as they come together, enzymes structure changes slightly
allosteric sites
distinct from the active site
binding to the allosteric site can induce a conformation change in the active site
is a mechanism of regulation
activates of inhibits reactions
cofactors
some enzymes require cofactors for optimal activity
can be inorganic materials like metal ions or organic compounds like NAD+
coenzymes briefly bind to enzyme but may be altered during the reaction
prosthetic groups are a metal or coenzyme that covalently bond but are not altered during reaction
enzyme calffication
oxidoreductases - transfer of oxygen or hydrogen atoms or electrons from one substrate to another
transferases - transfer of functional groups from one substrate to another
hydrolases - hydrolysis of a substrate
lyases - addition or removal of a group to form a double bond
isomerases - transfer of groups within a molecule
ligases - bond formation coupled with ATP hydrolysis
lysozyme
found in bodily secretions
antimicrobial agent
cleaves the peptidoglycan component of bacterial cells walls
leads to cell death
activation energy
enzymes speed up reaction, not altering amount of product formed
product formation levels off with time called equilibrium
this is a balance between forward reaction rate and reverse reaction rate
activation energy = amount of energy required to overcome energy barrier for reaction to take place
enzymes reduce amount of activation energy required
mechanisms to lower activation energy
catalysis by approximation - brining reactants closer makes an enzymatic reaction more likely
metal ion catalysis - metal ion is involved as cofactor
covalent catalysis - enzyme and substrate temporarily share electrons
acid-base catalysis - adding acid or base catalyses reaction
enzyme velocity
velocity = rate of reaction (umol/min)
it is the amount of substrate converted to product per unit of time
usually reported when time is 0 or before 10% of substrate is converted so the reaction is: fastest rate, highest substrate concentration, least number of products, least amount of feedback inhibition
concentrations
substrate concentrations affect initial velocity
1:1 ratio - doubling substrate doubles velocity
enzyme saturation = no further increase in velocity
doubling enzyme concentration doubles intial velocity
Vmax and Km
Vmax = maximum velocity at saturation point
half Vmax is when Michaelis constant is found known as Km
Km measures the enzymes affinity for a substrate and varies widely each enzyme
high Km = weak binding of enzyme and substrate
low Km = strong binding of enzyme and substrate so faster reaction
temperature and pH
rise in temp:
1. increase in thermal energy = overcome activation energy = increased rate of reaction
2. increase temp too much = beyond optimal temp = rapid decrease in reaction rate because multiple weak bonds break = alter active sit and denatures protein structure
small deviations in pH:
decreased activity
ionisation of groups in active site
large deviations in pH: denaturation
assays
assays = monitor the disappearance of substrate or appearance of product
can be measured by change in colour, change in light absorbance, chromatography, radiography
can be used to measure enzyme kinetics inc. Vo, saturation, Km, Vmax
substrate analogues
a chemical compound that is similar in structure to substate so it binds to active site
enzyme inhibition
inhibition = end product inhibits earlier pathway steps
prevents build up of unnecessary metabolites and energy use
often inhibiting product will bind to an allosteric site
activators do opposite
reversible inhibitors
bind to enzyme via weak non-covalent interactions (hydrogen/ionic bonds, hydrophobic interactions)
do NOT change the enzyme chemically
3 types: competitive inhibitors, non-competitive inhibitors, uncompetitive inhibitors
competitive inhibitors
similar shape/binding properties as substrate = some affinity for the active site
substrate competing for access to active site
therefore reduced rate of activity
can be overcome by increasing substrate concentration
Vmax remains unchanged but gets there slower so Km increases
non-competitive inhibition
inhibitors bind to allosteric site
conformational change of active site
substrate then cannot bind
dependent on concentration of the inhibitor - more inhibitors = less binding
Vmax is reduced, Km does not change
uncompetitive inhibition
inhibitor binds only to the enzyme/substrate complex
inhibits catalysis
Vmax and Km reduced
enzyme regulation of glycolysis
the enzyme hexokinase is allosterically inhibited by G-6-P (it’s product)
phosphofructokinase (PFK) is a key regulator of glycolysis - site of control
high ATP levels inhibit PKF - they allosterically bind to a region separate the the active site
inhibitors reduce the affinity of the enzyme for substrate
regulatory method: allosteric enzymes
multi-subunit enzymes = oligomeric (generally possesses a quaternary structure)
each subunit has own active site so there are multiple on one enzyme
cooperativity = binding of a substrate to active site causes conformational change and increases affinity for other active sites
regulated via allosteric sites
create sigmoidal curve = rapid increase increase in enzyme velocity over a narrow range of substrate concentration
allows allosteric enzymes to be sensitive to small changes in concentration of substrate
regulatory method: reversible covalent modifcation
addition or removal of phosphate groups onto or from an enzyme
changes tertiary structure to alter catalytic activity
can either activate or inhibit
protein kinases - addition of phosphate groups = phosphorylation
protein phosphatases - removal of phosphate groups = dephosphorylation
this method is rapid and reversible
regulatory method: proteolytic activation
zymogens or proenzymes are the name for the inactivated form of enzymes
zymogens activated by snipping the bonds between two or more amino acids (cleavage)
digestive enzymes produced in pancreas as zymogens
irreversible
enzyme synthesis
gene expression regulated enzyme synthesis
regulated at both gene transcription and protein translation stages
enzymes have short lived mRNA - control rate of protein expression
enzyme breakdown
enzymes also have a short half-life
most enzymes involved with important metabolic activities have short half lives - called labile enzymes
proteolytic degradation
