4 - Enzyme kinetics Flashcards
what are the facts you need to know about enzymes?
- they’re catalysts
- they’re specific
- they control well defined chemical reactions (they can be used in lab to investigate & develop a wide variety of diagnostic kits & therepeutic drugs)
what effect to enzymes can have major implications for disease?
small changes in their abundance, efficiency or distribution in tissue
what can enzymes catalytic behaviour be used for?
to diagnose disease (e.g. isoforms)
what are Vmax and Km used to describe?
they’re parameters to help describe how concentration of a substrate affects rate of catalysed reaction
what is Km?
equivalent to the substrate concentration where the initial reaction rate is half-maximal
(concentration of substrate which permits the enzyme to achieve half Vmax)
what is the point of max energy & least stability?
enzyme substrate complex at activation energy barrier -> point at which reaction could go either way (equillibrium)
what is Michaelis-Menten model used to explain?
explains the relationship between Vmax and Km & describes the rate of catalysis as a function of substrate concentration
how do you calculate Km?
k-1 + k2 / k1
is k-1?
backwards rate constant for enzyme dissociation from the substrate
what is k1?
the forward rate constant for enzyme association with the substrate.
what is k2?
the forward rate constant of enzyme conversion of substrate to product (P)
how are Vmax and Km measured?
-by measuring initial reaction velocity at V0 (at max - known - substratecapacity)
-then repeat at increasing substrate concentrations
what is Vmax?
a theoretical maximum - at infinite substrate concentration the initial reaction rate approaches Vmax
how can you accurately determine Vmax and Km?
by turning equation of V= Vmax[S] / Km +[S] into straight line equation y=mx+c
where is Vmax on Lineweaver Burk plot?
intersection of straight line with y - axis
where is Km on liineweaver Burk plot?
at intersection of straight line with x-axis
what do you use Vmax and Km for? (in simple terms)
to determine how good an enzyme is at doing it’s job (how fast it works)
what does a low Km value mean?
that enzyme only needs a little substrate to work at 1/2 max (so it’s efficient)
what does a high Km value mean?
that enzyme needs a lot of substrate to work at 1/2 max velocity (so less efficient)
do 2 different enzymes that have same Vmax always have same Km too?
NO - they could have same Vmax and different Km
how can you regulate different enzymes?
by activating different isozymes
what effect does competitive inhibiton have on Vmax and Km?
-Vmax does not change (as infinite substrate so substrates outcompete competitive inhibitors)
-Km varies (as depends on specific enzyme since substrate con 1/2 of max doesn’t necessarily outcompete number of competitive inhibitors, but it could)
what is effect on Vmax and Km in non-competitive inhibiton?
-Vmax varies (as inhibitor binds to different site so reaction rate not affected by substrate concentration)
-Km does not change (substrate not converted, not really affects)
what is feedback inhibition?
inhibition of rate limiting enzymes by end products - common mechanism of allosteric control
do allosteric enzymes follow Michaelis- Menten model?
No
what does increasing substrate concentration of allosteric enzymes result in on graph?
sigmoidal curve
what do allosteric factors affect enzymes?
modulate enzyme kinetic behaviour
what does lineweaver-Burk plot do?
enables accurate determination of Km and Vmax
what is orthosteric inhibition?
binding at same site (competitive)
how can allosteric enzymes be controlled?
by allosteric inhibitors and activators
competitive&non-competitive inhibitors are mostly reversible - but when are they irreversible?
some non competitive irreversible when involve formation or breakage of covalent bonds in enzyme complex
