lecture seven - enzyme kinetics Flashcards
what other types of intermediates are formed in an enzyme reaction, and why are these intermediates important?
transition states exist between ES intermediate and product
stability and rate constants (k1/k-1 and k2/k-2) determine whether a reaction will proceed
why is initial rate of enzyme activity (V0) determined at a very early time point in the reaction?
because after some time, the enzyme will become saturated with substrate, and that affects how fast the overall reaction proceeds
V0 is to measure the capability of the enzyme
why does a plot of V vs S taper off and eventually plateau at higher S levels?
at high S, M-M equation approaches a limiting value of Vmax
virtually all enzyme active sites are occupied by substrate once all sites are filled, and reaction cannot go faster
what is the Michaelis-Menten equation?
kinetic plot for generic enzyme involving one substrate
V = (Vmax * [S])/ ([S] + Km)
M-M equation terms
(V) is the rate of reaction at a given substrate concentration
(Vmax) is the fastest reaction rate possible under the given conditions
[S] is substrate concentration
Km reflects the enzyme affinity for substrate
why can the rate constant K-2 be ignored in the derivation of the M-M equation?
K-2 is rate-limiting and reduces the dissociation constant of the ES complex
why is a V vs S plot linear at low S concentrations?
M-M equation reduces to V = K(S)
why does V vs S plot curve at intermediate S values?
the whole M-M equation is used to describe reaction rate
why does V vs S plot plateau at high S levels?
there is no change in reaction rate once all the enzyme becomes almost fully saturated with substrate; an increase in substrate would not change anything
what does Km tell us about an enzyme?
tells us how efficient an enzme is at a low substrate concentration
low Km =
high Km =
low Km = enzyme is efficient at low substrate concentration
high Km = enzyme is inefficient at low concentration
what is the enzyme rate when [S] = Km?
the enzyme rate is 1/2 Vmax when S = Km
definition of turnover number
number of molecules of substrate that can be converted per second per molecule of enzyme
absolute value of turnover number for enzyme catalase?
40,000,000, still effective at high Km, which is rare
why doesn’t the turnover number of an enzyme change as the enzyme is purified?
turnover number is an intrinsic property
what is the Lineweaver-Burk equation?
alternate method of plotting kinetic data
1/v = (1/Vmax) + ((Km/Vmax) * (1/[S]))
reversible inhibitors
effects can be overcome by various emthods
irreversible inhibitors
effects cannot be overcome
non-competitive inhibitors
bind to a different site, away from active site, but the active site undergoes a conformational change which prevents the substrate from binding
competitive inhibitors
bind to an enzyme on its active site which keeps substrate from binding
competitive inhibitor effect on LWB plot
RAISES Km = -1/Km becomes less negative
1/V intercept stays the same = Vmax doesn’t change
LWB plot is a ___ plot
double reciprocal plot
** the inhibited reaction plot is higher than the un-inhibited reaction plot **
non-competitive inhibitor effect on LWB plot
make the line more steep
1/V changes indicating that the Vmax DOES CHANGE
1/[S] doesn’t change = Km doesn’t change either
mode of action of irreversible inhibitors
covalently modify a protein so that it cannot be reversed
what is specificity of action for TPCK
analog for reactive substrate chymotrypsin;
it is an affinity label and binds at the active site and reacts irreversibly with histidine residue at the active site of the enzyme
specificity of action of DIFP
DIFP is a group-specific irreversible inhibitor
irreversibly inhibits unusually reactive serine residues in the active sites of serine proteases
also modifies active site serine residue of acetylcholinesterase
specificity of action of iodoacetate
reacts with activated cystine residues in the active sites of various enzymes; acetamide residue forms a covalent bond with sulfur atom in active sites of enzyme
