2B- Enzymes and Clinical Enzymology

Question 1

Give a definition of “enzyme”

Answer 1

Enzymes are proteins that act like catalysts, which increase the rate of chemical reactions. They bind reactants/substrates, covert them to products, and release the products. They may be modified in the process but return to their original state after the reaction is complete. They also regulate metabolic processes in the body.

Question 2

What are the 3 steps in an enzyme-catalyzed reaction?

Answer 2

1. Binding of a substrate: E + S ES 2. Conversion of bound substrate into a bound product: ES EP 3. Release of product: EP E + P

Question 3

What is an active site?

Answer 3

It us usually a cleft or crevice in the enzyme formed by 1 or more regions of the polypeptide chain. In the site cofactors and other functional groups transform the bound substrate into products.

Question 4

Describe the “lock and key” model for substrate binding

Answer 4

Binding site is specific for a certain substate, and binds them through multiple hydrophocic interactions, electrostatic interactions or hydrogen bonds. Very specific.

Question 5

Describe the “induced-fit” model for substrate binding

Answer 5

As the substrate binds, enzymes undergo a conformational change that repositions the side chains of the AA’s in the active site and increases the numebr of binding interactions. Not a rigid “lock” like the lock-and-key model, but a dynamic fit created by the overall structure of the enzyme.

Question 6

What are coenzymes?

Answer 6

Coenzymes are complex nonprotein organic molecules that participate in catalysis by providing functional groups. They are usually synthesized by vitamins.

Question 7

How do metal ions contribute to the catalytic process?

Answer 7

Metal ions have a + charge and act like electrophiles (attract electron groups). They assist binding the substrate, or stabilize developing anions in the reaction. They can also accept and donate electrons in REDOX reactions

Question 8

How does pH affect enzyme activity?

Answer 8

Question 9

How does temperature affect enzyme activity?

Answer 9

Question 10

How do organophosphates work?

Answer 10

THey are covalent inhibitors- they form extremely tight bonds with the active site and inhibit them.

DFP, for example, forms an extremely tight bond with the active site of aceytlcholinesterase, thereby inhibiting the enzyme from degrading Ach in chemical synapses

Question 11

How does aspirin work?

Answer 11

Aspirin is a covalent inhbitor of the enzyme prostaglandin endoperoxide synthase (AKA cyco-oxygenase)

It resembles a protion of the prostaglandin precursor that is a physiological substrate for the enzyme

Question 12

How does penicillin work?

Answer 12

It is a covalent inhibior to the enzyme copoptidyl transferase

the beta-lactam ring of PCN has a strong resemblance of the noramal transpeptidation rection

it forms an irreversible inhibitor in the active site and are termed suicide inhibitors

Question 13

How does allopurinol work?

Answer 13

It is an covalent inhibitor that decreases gout by inhibing xanthine oxidase

the drug normally converts hypoxanthine to xanthine and xanthine to uric acid

Question 14

How does streptokinase work as a therapeutic agent?

Answer 14

It’s not a pure enzyme, but from a mixture of streptococcus

activates fibrinolytic enzyme system –> breaks down blood clots

can only be used once- elicits an immune response after the first use

Question 15

How does tissue plasminogen activator (t-PA) work as a therapeutic agent?

Answer 15

used to dissolve blood clots after a MI or CVA

has the same activity as streptokinase but because it’s basically the same as endrogenous t-PA, it doesnt have an immune response.

Question 16

How does asparaginase work as a therapeutic agent?

Answer 16

it breaks down asparagine (which is a preferred metabolic substrate for certain tumors)

used to treat adult leukemia

Question 17

What is the Michaelis-Menten equation?

Answer 17

Where K_m = (k₂ +k₃)/k₁

and V_max = k₃[E]

and

E + S ^—k1–>_<-k2– ES –k3–> E + P

Question 18

Interpret the graph (axises, [S], red line, Vmax/2, Km)

Answer 18

The x-axis is the substrate concentration, as you increase the [S] you increase the reaction velocity (v_i)

The red line is the enzyme showing MM kinetics

as you increase [S], the enzyme reaction velocity approaches Vmax but never reaches it, as all of the enzyme molecules contain bound substrate

Once it reaches Vmax, the enzyme is fully staurated.

1/2 of the Vmax is Km, which is where 1/2 of the enzymes contain bound substrate. The higher the Km, the higher the [S] required to reach 1/2Vmax

Question 19

What is the Km of MM kinetics?

Answer 19

It is the substrate concentration required to reach 1/2 Vmax

Question 20

What are 1 and 2 and 3?

Answer 20

1- uninhibited enzyme

2- competitively inhibited enzyme

3- noncompetitively inhibited enzyme

Question 21

What is compeitive inhibiton of an enzyme?

Answer 21

A competitive inhibitor competes with a S for the binding site

An increase in S can overcome the competitive inhbiiton

Km is increased beacuase they raise the [S] necessary to saturate the enzyme.

No effect on Vmax

Question 22

Label

Answer 22

The equation is the reciprocal of the MM equation, which is in the “y=mx +b” form. The picture of it is below.

1= y-axis, 1/Vi

2= slope, Km/Vmax

3= y-intercept, 1/Vmax

4= x-axis, 1/[S]

5= x-intercept, -1/Km

Question 23

What are noncompetive inhibitors and how do they affect enzyme kinetics?

Answer 23

Noncompetitive inhbiitors doesnt compete with a S for the binding site, but reduces the activity of the enzyme and binds equally well to the enzyme whether or not it has already bound the substrate.

The most common form is that the noncompetitive inhbitor binds to an allosteric site of the enzyme and changes the enzyme to work at a different velocity.

This is why the Vmax is changed but the rate, or Km is unchanged.

Question 24

What type of inhbition is shown in 1-3? Interpret why the changes occur.

Answer 24

1- competitive inhibiton. The slope and x-intercept change because they are Km/Vmax and -1/Km, respectively. The y-intercept is the same because that is 1/Vmax and that doesnt change in competitive inhbition.

2- mixed inhibition. There is change in both the Km and Vmax as the x and y intercepts change from normal and the slope changes as well.

3- noncompetitive inhbition. The x-intercept is constant, which is -1/Km, which makes sense with noncompetitive inhibition. However the slope and y-intercept change because they have Vmax in them, as they are Km/Vmax and 1/Vmax, respectively.

Question 25

What are allosteric activators and inhibitors?

Answer 25

Thy are compounds that bond to an allosteric site (another site on the enzyme that isnt the S binding site) and cause a conformational change that affects the affinity of the enzyme for the substrate.

Question 26

What is an allosteric enzyme?

Answer 26

they usually contain 2 or more subunits in which binding of 1 substrate to 1 subunit facilitates the binding of substrate to another subunit. For example, the first O2 bound to the first subunit of Hb has a little difficulty binding, but the conformational change of that 1 subunit makes it easier for the next subunit to bind to the next O2, which makes it easier for the 3rd and then of course the 4th.

Question 27

What is an allosteric activator? How does it affect Km?

Answer 27

The binding of an allosteric activator to an allosteric enzyme increases the affinity of the enzyme for the substrate. It decreases Km

Question 28

What is an allosteric inhibitor? How does it affect Km?

Answer 28

An allosteric inhibitor bind tightly to the T (taught) conformation, making it harder for the S to bind to the active site. Either [S] or [activator] must increase to overcome the inhibitor. It increases Km

Question 29

How does glycogen phosphorylase get activated? What is its use?

Answer 29

i. Muscle glycogen phosphorylase is the rate-limiting step in the pathway of glycogen degredation. It dregrades glycogen to glucose 1-phosphate. ii. It is regulated by AMP, which increases as ATP is used for muscular contraction. As you contract your muscle fibers AMP is released  activates glycogen phosphorylase  glycogen broken down so free glucose is used for energy. iii. It can also be activated by a phosphorylase kinase, which uses ATP  ADP to add a phosphate onto the enzyme. The glycogen phosphorylase kinase is activated when adrenaline is increased in the blood.

Question 30

How does PKA get activated and what does it do?

Answer 30

i. This enzyme phosphorylates a large # of enzymes that regulate different metabolic pathways. One of these is glycogen phosphorylase kinase (discussed above). ii. cAMP binds to PKA which dissociates and releases its activated catalytic subunits. These phosphorylate enzymes at serine residues.

Question 31

What is calmodulin?

Answer 31

i. Calmodulin is a modulator protein, which means it binds to other proteins and regulate their activity by caising a conformational change at the acatalytic site or by blocking the catalytic site. ii. Calmodulin binds to a number of different proteins and regulates their function. Once it binds 4 Ca++ ions, it activates enzymes.

Question 32

What is the pathway of Ca-calmodulin?

Answer 32

1. GTP binds to the G-protein  conformational change  binding of inactive target protein  binding activates the target protein  G-protein hydrolyzes a P from GTP to form GDP  changes conformation again  target protein dissociates which inactivates it  GDP is replaced by GTP and the process starts again

Question 33

What are zymogens?

Answer 33

G. There are some enzymes that are secreted from cells that are in the inactive form, called proenzymes. They have to undergo proteolytic cleavage to be activated. H. The precursors of proteases are called zymogens. They are synthesized in the inactive form because it prevents them from cleaving proteins before they should. That wouldn’t be good. Lol

Question 34

What is the mechanism of digestive enzymes secreted by the pancreas?

Answer 34

G. Chymotrypsinogen, a zymogen, is synthesized by pancreatic cells are secreted through ducts into the intestine. Once there, it is activated into chymotrypsin by the enzyme trypsin. That way chymotrypsinogen doesn’t start breaking down proteins until it reaches the intestine. H. Trypsin cleaves off a small peptide from chymotrypsinogen and makes it active.

Question 35

How can we use serum levels of enzymes to determine tissue damage?

Answer 35

G. When tissues are injured the enzymes inside of the cells are released into the blood. Measuring the levels of the enzymes gives a general sense of how damaged tissues are. H. Enzyme diagnosis is frequently useful in detecting diseases of liver, heart, pancreas, skeletal muscle, bone, prostate, blood cells.

Question 36

What diseases might be present with elevated AST and ALT?

Answer 36

liver and heart disease

Question 37

What diseases might be present with elevated amylase and lipase?

Answer 37

pancreatic diseases

Question 38

What diseases might be present with elevated H4 LDH?

Answer 38

heart and RBC

Question 39

What diseases might be present with elevated M4 LDH isozyme?

Answer 39

muscle and liver

Question 40

What diseases might be present with elevated CK-MB isozyme?

Answer 40

heart attacks after 6 hours

Question 41

What diseases might be present with elevated GGT?

Answer 41

liver disease, especially in biliary obstruction

Question 42

What are isoenzymes/isozymes?

Answer 42

multiple forms of an enzyme catalyzing the same reaction in the same organism, but often with different kinetics

Question 43

What is lactic dehydrogenase (LDH) and which isozymes are highest in myocardium?

Answer 43

It's an isozyme that converts pyruvate --\> lactate It is a isozyme tetromer with variable H and M subunits H₄ and H₃M are highest in myocardium

Question 44

What is creatine phosphokinase and which isozymes are highest in myocardium?

Answer 44

It's an enzyme that converts creatine --\> phosphocreatine in tissues 3 isozyme forms, MM (Skeletal muscle) MB (heart) and BB (brain) elevated MB is significant of a myocardial infarction

Question 45

What are troponins and how can they be used to Dx an MI?

Answer 45

they are muscle proteins, not enzymes Dx an MI within 4-6 hours, and persist in the serum for 2 weeks. use Ab tests to measure them

Question 46

Give the time sequence of appearance and disappearance of LDH, CK-MB, Mb, and Troponin I after an MI

Answer 46