Enzymes

Question 1

Transition state analogs

Answer 1

Usually man-made…simulate a real transition state and bind to a specific enxyme to prevent products from being made

Usually competitive inhibitors

Ex: HIV protease inhibitors and tamiflu

Question 2

Oxidoreductases

Answer 2

Catalysts in oxidation/reduction reactions

Glycolysis and krebs cycle

Question 3

Transferase

Answer 3

Transfer one functional group from one molecule to another molecule

Question 4

Hydrolases

Answer 4

Use water to cleave one molecule into two molecules

Question 5

Lyases

Answer 5

Work by adding or removing water, ammonia, or CO2 (but do not make 2 molecules from one)

Question 6

Isomerase

Answer 6

Intramolecular rearrangements

Question 7

Ligases

Answer 7

Catalyze high energy reaction using ATP…to join two molecules together

DNA ligase

Question 8

Initial velocity will increase linearly as…

Answer 8

Amount of enzyme is increased

Question 9

Initial velocity vs. [substrate]

Answer 9

Yields a bowed graph

Vmax = max velocity

Km = [subrate] at 1/2 of Vmax

Question 10

Lower the Km the ? The affinity of the substrate to bind to the enzyme…

Answer 10

Higher

Question 11

E + S (k1–>)(K2)E+P

Km =?

Answer 11

(K1+K2)/K3

Question 12

Michaelis - menten equation

Answer 12

Velocity = Vo = (Vmax*[S])/(Km+[S])

Question 13

At low [substrate]

What can be changed in MM equation

Answer 13

Adding [S] in denominator

Question 14

At high concentration Vo = ?

Answer 14

Vmax

Question 15

When [S]=Km

Then…

Answer 15

Vo=Vmax/2

Question 16

Kcat =

Answer 16

Constant that describes the limiting rate of any enzyme at substrate saturation

Kcat = Vmax/[total enzyme]

Value in sec(-1) units

Or…the amount of substrate that is turned into product for one enzyme molecule when it is saturated

Question 17

Regulation via non-covalent changes

Answer 17

Fastest form of regulation

Inhibit activity by REVERSIBLE binding to enzymes

Can either increase or decrease activity of an enzyme that is already there

Question 18

Regulation via covalent changes

Answer 18

Proteolytic processing, covalent modifications, irreversible inhibitors

Ex: phosphorylation/de-

Proteolytic processing = irreversible

Question 19

What is the MM plot NOT useful for?

Answer 19

Determining the type of inhibition

Question 20

Lineweaver-Burk PLot

Answer 20

Used to determine type of reversible inhibition is occuring

Question 21

LB plot

X-intercept

Y-intercept

Slope

Answer 21

-1/Km

1/Vmax

Km/Vmax

Question 22

Competitive inhibition

Answer 22

Binds at the active site

Can be reversed by….increasing [substrate]

Km increase as [I] increases = means to more inhibitor is added…the more substrate is needed to keep level of activity

Vmax = stays same!!! —> if enough substrate is added … it will overwhelm the effect of the inhibitor

**enzyme usually has a higher affinity for the inhibitor

Drugs: statins, methotrexate

Question 23

Mixed inhibition

Noncompetitive inhibition

Answer 23

Mixed = binds to a site distant from the active site to both E and ES

Km = increases
Vmax = decreases

Most ‘feedback inhibitors’ are these

Noncompetitve = special kind of mixed

Km = no change
Vmax = decreases

Bind to site distinct from active site and binds to either E or ES

Ex: organophosphate pesticides

Question 24

Uncompetitive inhibitors

Answer 24

Site distant from the active site

ONLY when the enzyme is in the ES complex

Both Km and Vmax are lowered

Question 25

Allosteric regulation

Answer 25

Important in metabolic pathways

Changes in one region/subunit of protein alter the conformation of other regions of subunits

Both activation and inhibition

Final products of pathway are often allosteric inhibitors of a key reaction in the pathway

S-shaped plot!!!

Always have multiple subunits

More likely to effect Km than Vmax

Question 26

Post-transitional modifications to proteins

Answer 26

Covalent modifications

Most imporant = phos, acetylation, ubiq

Question 27

Cholera toxin

Answer 27

ADP ribosylation

Permanently ADP-ribosylates an Arg. In the G-alpha subunit of the G-protein in the intestinal epithelial cells

—> results in cAMP production

—> secretion of water, Na+, K+, Cl-, HCO3- into the lumen —> dehydration

Caffeine = same effect but is a reversible inhibitor

Question 28

O-linked N-acetylglucosamine

Answer 28

Reversibly inhibits PK1 in response to hypoxia or other cellular stress in cancer cells (not in non-cancerous)

This will shuttle more glucose to the PPP

Where it can make DNA precursors and NADPH to protect cancer cells from oxidative stress…

Question 29

Prolyl and asparagunyl hydroxylation

Answer 29

Important in oxygen sensing by the transcription factor HIF

Tells the cell its becoming ischemic

Question 30

Proteolytic processing and zymogen activation

Answer 30

Breaking of bonds —> irreversible regulation

Common in digestion

Many proteins are made in an inactive form…zymogen

Then can be turned into active proteases once they arrive at desired destination via proteolytic cleavage

Question 31

Irreversible inhibitors

Answer 31

Different than transition staet analogs

= form covalent bond with substrate during catalyitc process which kills/inactiveates the enzymes

Question 32

Alpha-1-antitrypsin deficiency (A1AD)

Answer 32

May develop emphysema during their thirties even without smoking

Impair live function as well - most common cause for liver transplant in infants

Type of Serpin

Question 33

Antithrombin III

Answer 33

Also a serpin

Defects lead to increased formation of clots in veins and arteries