Enzyme Kinetics

Question 1

Why is the knowledge of enzyme kinetics useful

Answer 1

can quantify and enzymes speed, specificity (towards different substrates) and can be used to study how the activity of an enzyme is affected by other substances (like regulation via inhibitors)

Question 2

V naught

Answer 2

The initial velocity rate,

= the concentration of substrate disappearing// product produced per unit of time (mol/L*s)

Question 3

Why does the velocity of an enzyme decrease over time

Answer 3

Product inhibition can slow down the reaction, substrate decreases and thus the enzyme may no longer be saturated?

Question 4

First-order kinetics

Answer 4

rate is proportional to the substrate concentration

Question 5

Zero-order kinetics

Answer 5

rate is constant and independent of the substrate concentration

Question 6

Enzyme activity

Answer 6

measure of the quantity of active enzyme present and is thus dependent on conditions

the practical unit we use is U=enzyme units milimol/min`

Question 7

Specific activity

Answer 7

The activity of an enzyme per milligram of total protein

this help to give an indication of how pure an enzyme is

units= U/mg

Question 8

k1

Answer 8

rate of ES formation

Question 9

k2

Answer 9

rate of product formation

Question 10

k-1

Answer 10

rate of ES degradaaton

Question 11

When can k2 be ignored

Answer 11

during inital conditions, V naught

Question 12

Michealis-Menton equation assumptions

Answer 12

the equilibrium assumptions

Steady state assumptions

Question 13

The Equilibrium Assumption

Answer 13

assuume equilibrium between E and S and ES, such that, the equilibrium of formation and dissociation is only slightly disturbed by product formation

k2 &laquo_space;k-1

gives us the value Ks, the dissociation constant of the ES complex

this assumption is generally not correct lol

Question 14

Ks

Answer 14

The dissociation constant of the ES complex.

Tells us how tightly and enzyme binds its substrate

Question 15

Steady state assumption

Answer 15

assume that the ES complex is in steady state, thus the rate of formation of the ES complex equals that of the ES dissociaiton/ breakdown

Question 16

Km

Answer 16

Km is the Michaelis-Complex

under assumptions of equilibrium, Km=Ks, and is thus a good way to estimate Ks

Question 17

How can Km be used

Answer 17

can be thought of a smaller [S] to reach V50% has a greater affinity, thus the smaller the Km, the more specific/ tightly and enzyme can bind a given substrate

For example, glucose Km &laquo_space;Km for Fructose with the enzyme hexokinase. Thus Hexokinase has a higher affinity for glucose over fructose

Question 18

Kcat

Answer 18

Kcat when [S] -> infinity

Kcat is the turnover number when the enzyme is saturated

-> kcat= overall rate constant

Question 19

Kcat/Km

Answer 19

Used because the magnitude of Km is often equal to of slightly above the physiological concentration of substrates in pathway that operate at full capacity

And because kcat is defined at saturating levels of the enzyme ie [S]-> infinity

kcat/km is a good indicator of catalytic efficiency, in which the maximum value is 10^8/9 which is a perfect enzyme and means that every collision results in a reaction

rate at which enzyme and substrate collide

Question 20

Important of the Lineweaver-Burke Double Reciprocal Plot

Answer 20

Because it can be hard to get Vmax when the curve never reaches Vmax, then we cant get Km

Data may be linearized to find Km, however, our worst data contributes the most to the slope :(

still useful today for recognition of enzyme inhibition

Question 21

What factors does Enzyme concentration have on kinetics

Answer 21

increased enzyme concentration can increase the rate of the reaction, however it DOES NOT affect the Km

that is, the Km for any given enzyme is independent of enzyme concentration when enzyme concentration is limiting for the reaction. (ie// [S] is not limiting)

Question 22

Why may pH affect enzyme activity

Answer 22

Amino acids in the active site may need to be either protonated/ deprotonated in order to catalzyme the reaction

at super high or low pH enz could denature

Question 23

Why might Temperature affect enzyme activity

Answer 23

Increase of kineticv energy but at hight temp could denature

Question 24

Non-MM enzymes

Answer 24

Allosteric enzymes do not follow MM kinetics

-=> exhibit a sigmoidal binding curve (Unless conditions are adjusted)

Question 25

Regulation by availability of substrate or cofactor

Answer 25

recall that a low [s] ([s] ~km or less) Vo is linearly dependent upon [s]

if [s]&raquo_space; km then an increase in [s] will not affect Vo

need to consider [s] in subcellular compartments where the enzyme is located because [s] can vary between organelles