lecture seven - enzyme kinetics

Question 1

what other types of intermediates are formed in an enzyme reaction, and why are these intermediates important?

Answer 1

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

Question 2

why is initial rate of enzyme activity (V0) determined at a very early time point in the reaction?

Answer 2

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

Question 3

why does a plot of V vs S taper off and eventually plateau at higher S levels?

Answer 3

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

Question 4

what is the Michaelis-Menten equation?

Answer 4

kinetic plot for generic enzyme involving one substrate
V = (Vmax * [S])/ ([S] + Km)

Question 5

M-M equation terms

Answer 5

(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

Question 6

why can the rate constant K-2 be ignored in the derivation of the M-M equation?

Answer 6

K-2 is rate-limiting and reduces the dissociation constant of the ES complex

Question 7

why is a V vs S plot linear at low S concentrations?

Answer 7

M-M equation reduces to V = K(S)

Question 8

why does V vs S plot curve at intermediate S values?

Answer 8

the whole M-M equation is used to describe reaction rate

Question 9

why does V vs S plot plateau at high S levels?

Answer 9

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

Question 10

what does Km tell us about an enzyme?

Answer 10

tells us how efficient an enzme is at a low substrate concentration

Question 11

low Km =
high Km =

Answer 11

low Km = enzyme is efficient at low substrate concentration
high Km = enzyme is inefficient at low concentration

Question 12

what is the enzyme rate when [S] = Km?

Answer 12

the enzyme rate is 1/2 Vmax when S = Km

Question 13

definition of turnover number

Answer 13

number of molecules of substrate that can be converted per second per molecule of enzyme

Question 14

absolute value of turnover number for enzyme catalase?

Answer 14

40,000,000, still effective at high Km, which is rare

Question 15

why doesn’t the turnover number of an enzyme change as the enzyme is purified?

Answer 15

turnover number is an intrinsic property

Question 16

what is the Lineweaver-Burk equation?

Answer 16

alternate method of plotting kinetic data
1/v = (1/Vmax) + ((Km/Vmax) * (1/[S]))

Question 17

reversible inhibitors

Answer 17

effects can be overcome by various emthods

Question 18

irreversible inhibitors

Answer 18

effects cannot be overcome

Question 19

non-competitive inhibitors

Answer 19

bind to a different site, away from active site, but the active site undergoes a conformational change which prevents the substrate from binding

Question 20

competitive inhibitors

Answer 20

bind to an enzyme on its active site which keeps substrate from binding

Question 21

competitive inhibitor effect on LWB plot

Answer 21

RAISES Km = -1/Km becomes less negative
1/V intercept stays the same = Vmax doesn’t change

Question 22

LWB plot is a ___ plot

Answer 22

double reciprocal plot
** the inhibited reaction plot is higher than the un-inhibited reaction plot **

Question 23

non-competitive inhibitor effect on LWB plot

Answer 23

make the line more steep
1/V changes indicating that the Vmax DOES CHANGE
1/[S] doesn’t change = Km doesn’t change either

Question 24

mode of action of irreversible inhibitors

Answer 24

covalently modify a protein so that it cannot be reversed

Question 25

what is specificity of action for TPCK

Answer 25

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

Question 26

specificity of action of DIFP

Answer 26

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

Question 27

specificity of action of iodoacetate

Answer 27

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