Enzyme Kinetics

Question 1

When a cofactor or coenzyme is tightly or even covalently bound to an enzyme, it is called a….

Answer 1

prosthetic group

Question 2

Enzymes: covalent chemistry

Answer 2

a transient covelent bond forms between the enzyme and the substrate

Question 3

Enzymes: general acid-base chemistry

Answer 3

amino acid side chains of the enzyme can donate or accept protons to stabilize the transition state

Question 4

Enzymes: metal ion catlaysis

Answer 4

bound metal ions can help position teh substrate or can be powerful driving forces in redox reactions- almost 1/3 of all enzymes use a metal ion

Question 5

Km

Answer 5

The amount of substrate needed for the enzyme velocity to be at 1/2 vmax

Question 6

Kcat

Answer 6

The turnover number…
the number of substrate molecules converted to product in a given unit of time on a single enzyme molecule when the enzyme is saturated with substrate

Question 7

The ratio Kcat/Km

Answer 7

enzymatic efficiency

– the larger this number the “better” the enzyme

Question 8

Reversible inhibitors

Answer 8

Do not fundamentally alter the enzyme - act only so long as they are bound to the enzyme

Question 9

Competitive inhibitors:

Answer 9

Bind only to the E (not ES) and compete with the S for the AS

Question 10

What do competitive inhibitors do to the Km?

Answer 10

They raise it (because now you need more substrate to get to 1/2 vmax)

Question 11

Uncompetitive inhibitors

Answer 11

Bind to the enzyme in a place other than the active site and only to the (ES)!

Question 12

What do uncompetitive inhibitors do to the Km

Answer 12

change the apparent Km (lower it) because the slope does not change on the reciprocal plot (km/vmax), must lower Km by same factor as lower Vmax

Question 13

What do uncompetetive inhibitors do to the Vmax?

Answer 13

Lowers the Vmax - because they are altering the ES - you could think of it like making the ES tighter so less likely to release

Question 14

Mixed inhibitor

Answer 14

binds outside the active site, but can bind either E or ES

Question 15

Mixed inhibition effect on Km and Vmax?

Answer 15

Change both Km and Vmax

Question 16

Irreversible inhibitor

Answer 16

combine with or destroy a functional group on an enzyme that is essential for enzymes activity

Question 17

4 mechanisms of enzyme regulation

Answer 17

allosteric
covalent modification
binding of another regulator protein
proteolytic cleavage

Question 18

allosteric regulation

Answer 18

binding of another molecule changes the confirmation of teh enzyme and hence alters its function - often seen in feedback loop

Question 19

covalent modification - regulation

Answer 19

phosphorylation (and other modifications) are a powerful way to change the function of an enzyme. Many enzyme are constantly being P and de-P in response to intra or extra cellular signals or conditions

Question 20

Binding of another regulator protein - enzyme regulation

Answer 20

like allosteric regulation, small peptides can bind to enzyme and turn them on or off

Question 21

Proteolytic cleavage - enzyme regulation

Answer 21

some enzymes are inactive when they are first made (zymogen) but once cleaved into a smaller fragment (Generally by another protein) they become active

Question 22

What is the difference between a cofactor and coenzyme?

Answer 22

Coenzymes provide a chemical group for the reaction and are “used up”. Cofactors are not

Question 23

Coenzyme

Answer 23

small molecule bound in the enzyme that may provide a functional group

Question 24

Cofactor

Answer 24

usually metal ions bound by enzyme and used to help with chemistry

Question 25

Holoenzyme

Answer 25

enzyme + coenzyme or cofactor

Question 26

Apoenezyme

Answer 26

enzyme without coenzyme or cofactor

Question 27

How are enzymes named?

Answer 27

by what they do :)

Question 28

What determines the rate of a reaction

Answer 28

The activation energy

Question 29

Teachers opinion on lock and key vs induced fit

Answer 29

Induced fit - enzyme is complementary to the transition state (not the substrate)

Question 30

Binding favors the _________ state

Answer 30

transition

Question 31

enzymes can lower the activation energy by increasing the "Effective concentration" what does this mean

Answer 31

the confined space of the enzyme active site helps bring the substrates in close proximity so that positioned correctly

Question 32

If an enzymatic reaction results in the reduction of NAD to NADH ... NAD would be conisdered a_________

Answer 32

coenzyme

Question 33

If an enzymateic reaction required zinc, e.g. alcohol dehydrogenase, zinc would be considered ________________

Answer 33

a cofactor

Question 34

If we are using an enzyme for acid / base catalysis... it likely has what in the active sight

Answer 34

a.a. with a titratable hydrogen (pka near physiologic pH)

Question 35

Michaelis Mentin Equation

Answer 35

Vo = Vmax[S] / [S] + Km where Km = k-1 + k2 / k1

Question 36

Why is the Michaelis Mentin equation powerful?

Answer 36

We can use it to determine Km because When Vo = 1/2Vmax Km = [s]

Question 37

What is Km

Answer 37

The substrate concentration at which the enzyme is operating at 1/2 the maximal velocity

Question 38

Kcat is used to describe what?

Answer 38

The rate limiting step of a reaction (which will be the general rate constant)

Question 39

How are Kcat, Vmax, and [E] related?

Answer 39

The Kcat (or turnover number) is equal to the maximum velocity divided by the enzyme concentration Kcat = Vmax / E Which makes sense because Vmax is the maximal velocity of the reaction at a given enzyme concentration...if we then divide that by by the enzyme concentration, we will get the Kcat, which is the number of molecules being made per second by a single enzyme We could also write it as Vmax = Kcat[E]

Question 40

What does a high Kcat mean?

Answer 40

A high Kcat means that one enzyme is able to turnover muchos molecules per unit time

Question 41

Kcat/Km usefullness

Answer 41

General description of enzymatic efficiency... takes into account both the speed of the enzyme (Kcat) and how much substrate you need to supply to get to the enzyme (Km)

Question 42

What limits enzyme

Answer 42

Diffusion of substrate around it into the active site (usually around 10^9 kcat/km) - the only thing slowing them down is how rapidly substrate is diffusing to enzyme

Question 43

Why is Michaelis Menten useful?

Answer 43

Helps us sort out the mechanism of complex enzymatic reactions

Question 44

What are the 3 types of reversible inhibitors?

Answer 44

comp. uncomp. mixed.

Question 45

what is the utility of Lineweaver Burke?

Answer 45

Allows us to evaluate inhibitors - determine what type

Question 46

Competetive inhibitor effect of Lineweaver Burke plot with increasing [i]

Answer 46

Increases slope (km/vmax) without changing vmax (y int)

Question 47

Competetive inhibitor effect on Kcat

Answer 47

Does not change Kcat because Kcat = Vmax/[e] and we are not changing Vmax

Question 48

Uncompetitive inhibitors ... which parameters do they effect?

Answer 48

They effect all parameters Kcat Km and Vmax However, note that they are effecting Km and Vmax by the same magnitude - lowering Km - which means they are lowering the [s] at 1/2 vmax... which is likely because we lowered vmax because we are effectively taking out enzymes - which means that even though our affinity is the same we have lowered vmax - plus we have also lowered kcat because kcat = Vmax/[e]... we lowered Vmax but we have not in reality lowered our [e], only our effective [e] and thus, we have also lowered our kcat

Question 49

Mixed inhibitors usually affect which parameters? effect on Lineweaver Burk?

Answer 49

We are bloody chaging everything Our slope (km/vmax) is increasing Our x intercept is increasing (becoming less negative) (-1/km) Our y intercept is increasing (alpha/vmax - so vmax is decreasing) Thus, our Km is increasing, our vmax is decreasing, and our slope is increasing (km/vmax)

Question 50

Where do irreversible inhibitors usually act?

Answer 50

The active site

Question 51

In metabolic cascades, which enzyme do we usually want to target via negative feedback

Answer 51

Usually the first

Question 52

We can also have enhancers that activate enzyme

Answer 52

allosteric regulation to increase enzyme activity

Question 53

Our covalent modifcations generally reversible?

Answer 53

Yes - turn on / off

Question 54

Enzymatic regulation via proteolytic cleavage is common where?

Answer 54

digestive system | e.g. trypsinogen --> trypsin