Steele - Lecture 3

Question 0

What is the difference between an allozyme an isozyme?

Answer 0

An allozyme is a form of a protein encoded by different alleles (maternal, paternal) - “allelic enzyme”

An isozyme is two forms of a protein encoded by different genes but catalyze the same reaction (albeit at different kinetics)

Question 1

How can variant hemoglobin be identified?

Answer 1

Through gel electrophoresis

Question 2

What are 3 three isozymes of creatine kinase and what are the subunits?

Answer 2

CK1- BB
CK2- MB (heart)
CK3- MM (skeletal muscle)

Question 3

How can you detect injuries with creatine kinase

Answer 3

CK2 elevation: heart damage

CK3 elevation: skeletal muscle damage

Question 4

What is perhaps a better marker for heart damage than CK-MB?

Answer 4

Cardiac troponin- greater specificity, elevated over longer period than CK2

Question 5

List 4 properties of protein modification:

Answer 5

1) found in virtually all eukaryotic proteins
2) modification related to protein localization
3) can be reversible or irreversible
4) sometimes catalyzed by enzyme, sometimes not

Question 6

What are two categories of N-terminal modification of proteins? Give some examples

Answer 6

1) proteolysis: exoprotease: removal of initiator met, endoprotease: removal of signal peptide sequence
2) amino-terminus modification: acetylation, myristoylation

Question 7

What is the purpose of N-terminal acetylation?

Answer 7

stability against degradation by aminopeptidases

prevents glycation at amino-terminus by reducing sugars

Question 8

How is n-terminal acetylation achieved?

Answer 8

methionine aminopeptidase cleaves off initiator methionine,

acetylase + acetyl CoA then acetylates the N-terminus

Question 9

What is a useful clinical tool related to protein glycation?

Answer 9

High levels of glucose in blood ends up glycating an n-terminal Valine on beta chain via a NON ENZYMATIC REACTION

This can be measured in high levels in diabetics as HbA1c

Question 10

What are the most phosphorylated amino acid residues. which are the least?

Answer 10

Serine: Threonine: Tyrosine 1800:200:1

~1/3 of all proteins contain at least 1 phosphate

Transient. ~2% of human genome encodes kinases and phosphatases

Question 11

Describe the pathway for inflammatory response TF NFkB

Answer 11

IkB asks NLS on IFkB. IKK phosphorylates IkB in response to extracellular signal, allowing NFkB to localize to nucleus and effect response.

Question 12

What kind of reaction removes a phosphate group from an AA residue?

Answer 12

phosphatase reaction (involves hydroylsis)

Question 13

How is protein kinase A activity mediated?

Answer 13

4 cAMP binds to the two regulatory subunits (R) of PKA), revealing the active sites on the two catalytic subunits (C)

Question 14

What is especially interesting about the insulin kinase cascde?

Answer 14

Self-Phosphorylation of insulin receptor which then binds IRS-1

Question 15

How are proteins kept inactive? How are they activated?

Answer 15

They are translated in a form called “pro-“ex: pro-insulin

Subject to SPECIFIC (as opposed to general proteases for degradation) protease activity that cleaves them into their active form, most often in secretory vesicles and occasionally extracellulary

Question 16

How is protein destruction achieved?

Answer 16

Tagged generally with ubiquitin, degradation by a large protein complex called proteasome

Question 17

In the ubiquitination pathway, what protein is responsible for specificity of the pathway?

Answer 17

E3 (>700 identified), increases specificity for specific targets

Question 18

Plasma from patients with multiple myeloma display a strong band on an electrophoresis gel hat’s not present in normal individuals. What does this band indicate?

Answer 18

Excess production of a certain immunoglobulin protein

Question 19

How is multiple myeloma treated?

Answer 19

Velcade- proteosome inhibitor. Uncertain how it works but somehow allows multiple myeloma cells to be killed more easily.

Question 20

What is the structure of hemoglobin

Answer 20

Heterotetramer composed of heme + 4 globulin polypeptides

Adult: 2 alpha 2 beta
Fetal: 2 alpha 2 gamma

Heme = Iron- protoporphyrin IX

Question 21

Where is the iron relative to the plane of the protoporphoryn ring?

Answer 21

Iron lies just outside of the plane of the ring, pulled there by a histidine. Binding of oxygen to iron moves the ring up into the plane slightly.

Question 22

What are the two states of hemoglobin?

Answer 22

T state = tuat = low affinity for Oxygen = deoxy state

R state = relaxed = high affinity for oxygen = oxy state

Question 23

Binding of oxygen to hemoglobin becomes progressively easier. How?

Answer 23

Positive cooperativity due to conformational changes of subunits

Question 24

What extraneous factors reduce affinity of oxygen to adult hemoglobin?

Answer 24

1) Increase in acidity (Bohr effect)
2) Increase in carbon dioxide
3) Increase in temperature
4) Increase in 2,3-BPG (a single molecule binds one hemoglobin!)
5) Increase in fetal hemoglobin (by mixing with maternal blood) - HbF has higher affinity of oxygen than HbA

These all stabilize the T-state

Question 25

How do RNA’s catalyze reactions? How DON’T they catalyze reactions?

Answer 25

Lower activation energy of reaction (increasing kinetic rate)

They DO NOT change Keq or free energy change. They do not make nonspontaneous reactions (+ delta G) to spontaneous reactions (- delta G)

Question 26

How is Vo affected by substrate concentration?

Answer 26

Vo increases with increasing substrate concentration, until the solution becomes saturated with substrate and the reaction reaches Vmax

Question 27

What is Km of an enzyme? What does a high Km signify?

Answer 27

Km is the amount of substrate needed to reach 1/2 Vmax.

A high Km therefore indicates low affinity for enzyme-substrate.

Question 28

What is the major assumption of the Michaelis-Menton equation?

Answer 28

Assume that back reaction ES–>E+S is negligible (by using Vo)