Biochemistry Exam 2: Enzyme Kinetics

Front 1

What did early work of enzyme kinetics involve?

Back 1

crude preps, only observe reaction of reactants to products

Front 2

What eventually were we able to do with enzyme kinetics?

Back 2

Measure rates

Front 3

In the presence of ________ and _________, enzyme kinetics became a powerful tool.

Back 3

Competitors and Inhibitors

Front 4

For chemical kinetics where A->P, at constant T what is rate?

Back 4

Frequency of A colliding

Front 5

What is proportionality constant for chemical kinetics?

Back 5

Rate constant k

Front 6

In chemical kinetics A -> P, what is instantaneous appearance of P or disappearance of A called?

Back 6

velocity v; v=d[P]/dt = -d[A]/dt = k[A]

Front 7

What are units of velocity and rate constant?

Back 7

Velocity is M/sec; rate constant is 1/sec

Front 8

What does order of reaction correspond to?

Back 8

Molecularity - number of reactants that must collide to form P

Front 9

1ST ORDER IS WHAT TYPE OF MOLECULARITY?

Back 9

Unimolecular reaction

Front 10

What is velocity and order nad molecularity of 2A -> P?

Back 10

bimolecular 2nd order; v = d[A]/dt = k[A]^2

Front 11

What are units for rate constant k in second order reaction?

Back 11

M^-1sec^-1

Front 12

What is v or instantaneous velocity for A + B -> P?

Back 12

k[A][B]

Front 13

How do we know that A + B -> P is second order?

Back 13

Because first order in A and first order in B = 2nd order

Front 14

Why are higher order reaction such as termolecular uncommon?

Back 14

Because must rely on simultaneous collision of 3 or more molecules

Front 15

What does rate equation describe?

Back 15

Progress as a function of time

Front 16

What is rate equation for 1st order reaction A -> P?

Back 16

ln[A] = ln[A]0 - kt

Front 17

What is the graph look like for rate equation of 1st order reaction such as A-> P?

Back 17

plot of ln[A] as y axis and time as x axis and straight line with slope -k

Front 18

For first order reactions such as A-> P, time for half A to decompose is _______.

Back 18

Half life

Front 19

Half life of A in first order reaction A->P is constant and independent of ________.

Back 19

[A]o

Front 20

What is the formula for half life in first order reaction?

Back 20

t1/2 = .693/k

Front 21

What is reaction equation for second order reaction with 1 type reactant? so 2A - > P

Back 21

1/[A] = 1/[A]o + kt

Front 22

What is half life of second order single type reactant dependent on?

Back 22

[A]o

Front 23

What is the equation for half life of second order single type reactant?

Back 23

t1/2 = 1/ k[A]o

Front 24

How do we distinguish first order from second order reaction plot?

Back 24

1st order - ln[A] vs. t
2nd order - 1/[A] vs. t

Front 25

For second order reaction: A + B -> P in which B concentration much greater than A what does rate depend on? Why?

Back 25

Concentration of A since in limited supply

Front 26

E + S ⇄ ES → P + E, When S is high enough to convert all E to ES, what is rate limiting step?

Back 26

Second Step

Front 27

E + S ⇄ ES → P + E, When S is high enough to convert all E to ES, the overall reaction is still sensitive to S concentration. T/F

Back 27

False, it is insensitive to S concentration

Front 28

E + S ⇄ ES → P + E, When S is high enough to convert all E to ES. Which steps are reversible and irreversible?

Back 28

K1 reversible and K2 irreversible

Front 29

What is Michaelis – Menten Equation: with respect to E + S ⇄ ES → P + E?

Back 29

It describes rate of enzymatic reaction as function of S

Front 30

What is rate of P formation in E + S ⇄ ES → P + E?

Back 30

v = k2[ES]

Front 31

What is rate of production of ES in E + S ⇄ ES → P + E?

Back 31

Rate of production of ES is Δ between rates of appearance and disappearance
d[ES]/dt = k1[E][S] – k-1[ES] – k2[ES]

Front 32

What are the two assumptions necessary to integrate the Michaelis-Menten Equation with E + S ⇄ ES → P + E?

Back 32

1. Assumption of equilibrium in that k-1 » k2 so first step reaches equilibrium Ks = k-1/k1 = [E][S]/[ES] where ks is dissociation constant of 1st step
2. Assumption of steady state: under common physiological conditions [S] » [E]; with exception of first millisec [E] and [ES] remain ≈ constant until [S] exhausted

Front 33

In E + S ⇄ ES → P + E, ES maintains what state and how is it treated?

Back 33

Steady state and constant value

Front 34

Who first proposed the steady state assumption?

Back 34

GE Briggs and JBS Haldane

Front 35

How are kinetic expressions useful?

Back 35

Experimentally measurable

Front 36

_________ and __________ are not measurable in E + S ⇄ ES → P + E. But ___ is measurable.

Back 36

ES and E; Et = ES + E

Front 37

E + S ⇄ ES → P + E; What is rate equation for overall reaction as function of E and S?

Back 37

k1[E][S] = k-1[ES] + k2[ES]

Front 38

E + S ⇄ ES → P + E; How can you rearrange this equation to use only K's and give Michaelis Constant Km?

Back 38

Km = (k-1 +k2)/k1 or KM[ES] = ([E]T – [ES])/[S]

Front 39

In Michaelis Menten Equation, using vo rather than v minimizes some complications such as ....

Back 39

Reversible reactions
Product inhibition of E
Progressive inactivation of E

Front 40

How do we meet steady state conditions in Michaelis Menten Equation for E + S ⇄ ES → P + E?

Back 40

Concentration of S has to be greater than E so that E can repeatedly bind to S and convert to P thus ES is constant

Front 41

What is maximum velocity in E + S ⇄ ES → P + E?

Back 41

It is when there is increase in S concentration so E is saturated all in ES form. Vmax = k2[E]

Front 42

Combining all of the steps what is the full equation (End equation) for Michaelis Mentin Equation?

Back 42

v0 = Vmax[S]/(Km + [S])

Front 43

When does Km = [S]?

Back 43

When Vo = 1/2Vmax

Front 44

What is Km?

Back 44

It is S concentration term and measure of catalytic efficiency.

Front 45

What does small Km and large Km mean?

Back 45

Small Km means Vmax at lower [S] or higher catalytic efficiency; large Km means Vmax at higher [S] or lower catalytic efficiency

Front 46

T/F Km is the same for every enzyme substrate pair.

Back 46

False it is unique for everyone enzyme substrate pair.

Front 47

Km is also a function of ________ and ________ while being unique for every enzyme substrate pair.

Back 47

Temperature and pH

Front 48

What is Ks= ? and what does it mean?

Back 48

k-1/k1 ; it is the dissociation constant of Km

Front 49

What happens when Ks increases or decreases?

Back 49

As Ks increases E's affinity for S decreases and As Ks decreases, E's affinity for S increases

Front 50

Km can be a measure for E's affinity for S. If Km high ... and if Km low...

Back 50

High Km - low affinity for S
Low Km - high affinity for S

Front 51

What is the catalytic constant or turnover number?

Back 51

Number of reactions/active site/ unit of time

Front 52

What is the formula for the catalytic constant?

Back 52

Kcat = Vmax/[E]t

Front 53

What does it mean if Kcat is high and if Kcat is low?

Back 53

Kcat high - fast turnovers, fast enzyme

Kcat low - low turnovers, slow enzyme reaction

Front 54

Even thought Kcat is constant, Vmax is approximately equal to ____.

Back 54

[E]t

Front 55

In a simple E equation Kcat = ?

Back 55

k2

Front 56

What happens when [S] << Km

Back 56

Little ES forms and [E] = [E]t

Front 57

In determining Vmax in Vo vs. [S] plot, what is Vo when [S] = 10Km? Also why is it difficult to determeine Vmax from this plot?

Back 57

Vo = about 91% of Vmax
Vmax is often underestimated by this plot

Front 58

What did Hans Lineweaver and Dean Burk formulate?

Back 58

They formulated a better way of calculating Vmax by using reciprocal of Michaelis Menten equation. -> 1/Vo = (Km/Vmax)(1/[S]) + (1/Vmax)

Front 59

What is a disadvantage of Lineweaver Burk Plot?

Back 59

There is a crowding of data along vertical axis

Front 60

What do small errors in [S] and Vo lead to in Lineweaver Burk Plot Data collection?

Back 60

Large errors in 1/Vo -> large errors in Km and Vmax

Front 61

T/F Steady state kinetics can establish reaction mechanisms.

Back 61

False

Front 62

What happens if kinetic data does not match given mechanism?

Back 62

Mechanism must be rejected

Front 63

What kind of insight to steady state kinetics provide for ES?

Back 63

Provides insight into buildup and breakdown of ES, provides little information on nature of ES, Can not provide information on number of intermediates of ES

Front 64

What are bisubstrate reactions and how much of chemical reactions to they make?

Back 64

60% of chemical reactions are bisubstrate reactions; bisubstrate - 2 substrates and 2 products

Front 65

What kind of reactions are bisubstrate ones usually?

Back 65

Transfer Reactions or Oxidation Reduction Reactions

Front 66

What are two types of Bisubstrate reactions?

Back 66

Sequential Reactions - all S’s bind with E before reaction occurs and P’s released

Ping Pong reactions: group transfer reactions where 1 or more P’s released before all S’s bound

Front 67

What is the ordered mechanism of a sequential reaction?

Back 67

Binding of E to S required before it can open second binding site.

Front 68

What is random mechanism to sequential reactions?

Back 68

No specific order of S binding , both binding sites are available

Front 69

What are Enzyme inhibitors?

Back 69

They can alter Enzyme activity by altering S binding or P release

Front 70

What are irreversible inhibitors?

Back 70

They bind so tightly that they block enzyme activity completely

Front 71

What is competitive inhibition?

Back 71

Ihibition where I competes for E binding site.

Front 72

Who do competitive inhibitors compete with for E binding site?

Back 72

S they usually look like S as well

Front 73

What is one of the physiological methods of E control?

Back 73

Competitive inhibition by Product P

Front 74

How do Transition state analogs function as good inhibitors?

Back 74

They mimic TS

Front 75

What does degree of competitive inhibition depend on?

Back 75

Fraction of E that was bound to Inhibitor

Front 76

What is an uncompetitive inhibitor?

Back 76

Where I binds directly to ES complex and not E; doesn't need to resemble S; distorts E active site rendering it catalytically inactive

Front 77

What is mixed inhibition?

Back 77

Inhibitors bind both E and ES complex; may be reversible, affect S binding, affect catalytic activity, Binds to E sites participating in both catalysis and S binding

Front 78

What is an example of a good mixed inhibitor?

Back 78

Metal Ions

Front 79

What is another name of mixed inhibitors?

Back 79

Noncompetitive inhibitors

Front 80

How do organisms regulate and coordinate metabolism?

Back 80

Control enzyme availability - amount of E depends on synthesis and degradation; and enzyme activity - controlled by product inhibition - feed back

Front 81

What is allosteric control of enzyme activity?

Back 81

Binding at site other than active site to change conformation

Front 82

What is covalent modification of E?

Back 82

Phosphorylation/Dephosphorylation of E; catalyzed by protein kinases and protein phosphates

Front 83

_______ of human protein participating in biological processes is subject to covalent modification,

Back 83

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