Lecture 2.4: Enzyme Kinetics

What does rate (V) equal

Rate (V) = K[S]

What is Vmax

The theoretical maximum rate at which an enzyme can catalyze a reaction when the active sites are completely saturated

What is the michealis-menten kinetics equation

V0 = Vmax [S] / Km + [S]

What does increasing [E] do to the Vmax of a reaction

Increases the Vmax

Define Km?

The amount of substrate needed to reach ½ Vmax

What does a low Km tell you about the enzyme?

Low Km = enzyme has high affinity for substrate

What does a high Km tell you about the enzyme?

High Km = enzyme has high affinity for substrate

In the case of Hexokinase (brain) and Glucokinase (liver) why is it important that Hexokinase has a very low Km?

Low Km means high affinity which in this case is important for the brain to be able to absorb glucose from the blood even if glucose concentration are very low The brain primarily runs of glucose for energy.

What do the Km values show for the substartes associated with the eznyme Chymotryspin?

N-Benzoyltyrosinmide is much more preferred as a substrate. This can be seen by the low Km (high affinity)

What is the Michealis-Menten equation when Km is much greater than [S]

V0 = [S]

Why doe Glycyltyrosinylglycine have such a high Km

The two glycine amino acids make the substrate very flexible and unfavorable to lock into a strict conformation in the active site

(T/F) Km is effected by [E]

False, enzyme concentration does not effect Km

What is the Michealis-Menten equation when Km is much less than [S]

V0 = Vmax

In a lineweaver-burk plot what is the Y-axis

1 / V0

In a lineweaver-burk plot what is the X-axis

1 / [S]

In a lineweaver-burk plot what is the Y-intercept

1 / Vmax

In a lineweaver-burk plot what is the X-intercept

-1 / Km

In a lineweaver-burk plot what is the slope

Km / Vmax

Define Kcat

amount of turnover for one enzyme at full saturation

What does a high Kcat mean

Faster turnover

What is the equation for Kcat

Kcat = Vmax /[E]_T (Units: Sec^-1)

Define catalytic efficiency (specificity constant)

The amount of product turnover at a specific concentration

What is the equation for the specificity constant

Kcat / Km

What does a bigger specificity constant mean

Bigger specificity constant = better enzymes

What is the upper limit for Kcat / Km (Catalytic Perfection)

Between 10⁸ - 10⁹

On a plot of ​[product] versus time for an enzyme-catalyzed reaction, the V₀ is equal to the:

a) slope of the line / ​[S].

b) [P] at the lowest time point.

c) slope of the line.

d) y-intercept.

e) x-intercept

a) slope of the line / ​[S]

What is the same of this graph

Hyperbolic

Which of the following is NOT TRUE about the graph of  V₀ vs initial ​[S]?

a) Initial velocity can be experimentally obtained by measuring the concentration of product formed early in the reaction. 

b) At low substrate concentration ​[S], initial velocity increases as ​[S] increases. 

c) Enzyme concentration ​[E] is kept constant. 

d) At high substrate concentration ​[S], initial velocity increases as ​[S] increases.

d) At high substrate concentration ​[S], initial velocity increases as ​[S] increases.

In Michaelis-Menten kinetics, k₁ is a constant that reflects _______, whereas k₂ is a constant that reflects  _______.

the rate of ES formation; the rate of product formation

In Michaelis-Menten kinetics, under saturating conditions, _______ is relatively constant.

V₀

(T/F): For a given enzyme, the maximum velocity remains constant regardless of enzyme concentration. 

Flase

K_M is equal to:

[S] when V₀ = ½ V_max

What does each letter represent

A = Vmax

B = ½ Vmax (high [E])

C = ½ Vmax (low [E])

D = Km

Fill in the Blanks: Brain hexokinase has a (higher/lower)K_m than liver glucokinase, which means it reaches ½ V_max at a (higher/lower) ​[S].  This is physiologically relevant because it means that brain hexokinase works (better/worse) than liver glucokinase

lower; lower; better

(T/F) For a given enzyme, increasing the concentration of enzyme will increase Km.

False

Which of the following is the Michaelis Menten Equation?

a) V_max = V₀​[S] / (K_m + ​[S])

b) V₀ = V_max​[S] / (K_m + ​[S])

c) K_m = V_max​[S] / (V₀ + ​[S])

d) ​[S] = V_max​[V₀] / (K_m + ​[S])

b) V₀ = V_max​[S] / (K_m + ​[S])

A plot of 1 / V₀ versus 1/​[S] is called a ________ plot. Data in this plot have a slope equal to ________ and a y-intercept equal to _______

lineweaver-burk; K_m / V_max; 1 / V_max

Which of the following enzymes would be considered very highly efficient/near catalytic perfection? Select all that apply.

a) carbonic anhydrase

b) acetylcholinesterase

e) superoxide dismutase

Fill in the Blanks

A =

B =

C =

D =

A = V0

B = Vmax

C = [S]

D = Km

An enzyme-catalyzed reaction has a Km of 1 mM and a Vmax of 5 nM/sec. What is the reaction velocity when the substrate concentration is 0.25mM? Give your answer as a number only in nM/sec.

1.0

An enzyme that follows Michaelis-Menten kinetics has a initial velocity of 300 nM/s at a substrate concentration of 30 μM. The maximum velocity is 400 nM/sec. What is the Km for this enzyme in μM? Give your answer as a number only.

10

Estimate the Km

30 mM

An enzyme's initial velocity was measured at different substrate concentrations and placed into a Lineweaver-Burk plot. The linear regression of the data yielded the following equation: y = 0.484x + 0.195

What is the Km of the enzyme? Round to 2 decimal places, and do not include units in your answer.

Y-int = 1/ Vmax

0.195 = 1/ Vmax

Vmax = 1/ 0.195 —> 5.128

Slope = Km / Vmax

0.484 = Km / 5.128

Km = .484 × 5.128 —> 2.48

Although graphical methods are available for accurate determination of the Vmax and Km of an enzyme-catalyzed reaction, sometimes these quantities can be quickly estimated by inspecting values of V0 at increasing ​[S]. Without making a graph, estimate the Vmax and Km of the enzyme-catalyzed reaction for which the data were obtained.

Vmax = _____ μM/min

Km = _____ μM

Vmax = 140 μM/min

Km = 10 μM — Inspection of the table indicates that this Vo occurs at ​[S] = 1 x 10^-5 M, thus Km ≈ 1 x 10^-5 M or 10 μM.

What is the difference between reaction rate and turnover number?

Reaction rate is how quickly the substrate can be turned into product (speed)

The turnover number is maximum number of substrate molecules a single enzyme active site can convert into product molecules per unit of time (efficiency)

Carbonic anhydrase is a vital enzyme that maintains the buffering capacity of blood. It can use two substrates,

CO₂ or HCO₃- ​ Using the table below, which would be the best choice for a substrate and why? ​

CO2; the specificity constant is higher​ (Kcat/Km)

What is the specificity constant of ribonuclease for cytidine cyclic phosphate?

100,000

Define the equation velocity of an uncatalyzed chemical reaction

v = k[S]

Why is it better to use a Lineweaver–Burk plot than a Michaelis–Menten plot

Lineweaver-Burk plots allow for a more precise measurement of the values and express the data as a linear relationship

Explain how V_max and K_m from a Lineweaver-Burk plot.

Vmax:

• Find the y-intercept — the point where the line crosses the y-axis (where 1/[S] = 0)

• This intercept equals 1 / Vmax

• So: Vmax = 1 / (y-intercept)

Km:

• Find the x-intercept — the point where the line crosses the x-axis (where 1/v₀ = 0)

• This intercept equals −1/Km

• So: Km = −1 / (x-intercept)

What is the BEST measurement for analyzing enzyme efficiency

Specificity constant (Kcat / Km)

What is the slope equation for a Michealis-Menten plot

Slope = V0 / [S]