Biochem Mechanisms for Regulating Metabolism (LSK)

Question 1

Protein-ligand binding

Answer 1

The affinities for each successive ligand is different, but the pathways from start to finish are energetically equal

Question 2

Cooperativity: features, examples, and binding curve (+, -)

Answer 2

Identical binding sites
Usually on identical subunits of homo-oligomers OR on the same subunit

Identical ligand (X)

Ex. Hemoglobin: identical binding sites for O2 (repeated on each subunit); produce same signals in lab

Effects on binding curve
If we could see separate curves (but we can’t because identicalness produces same signal in lab), we’d see curves like in allostery. But because we can’t measure separately, we end up with a weighted average curve, leading to much steeper binding.
SLOPE >1 = HALLMARK

Binding affinities are not identical
Positive: second ligand binds tighter than the first
Negative: second ligand binds weaker than the first

Question 3

Allostery: features, examples in nature, and binding curve

Answer 3

Two different binding sites
Could be on two different subunits or on two different places on the same subunit

Different ligands (X, Y)

Ex. Metabolism: changing nutrients allow reaction rates to be turned up or down
DNA transcription: can change transcriptome in response to environment

Effects on binding curve
Can have a positive or negative effect (binding X could make Y bind weaker or stronger)
Shift in Kd right or left depending on whether it’s inhibitory or enhancing

Question 4

Function, tissue distribution, and regulation of GLUT isoforms

Answer 4

s

Question 5

Km values to physiological concentrations of ligand

Answer 5

Km = concentration of substrate that allows enzyme to be at half of maximal velocity

Question 6

Kd values to physiological concentrations of ligand

Answer 6

Kd = the concentration at which 50% of binding sites (receptors) are occupied by the drug

Question 7

Mechanisms for altering substrate/metabolite concentration and consequence on enzyme activity

Answer 7

s

Question 8

Strategies for allosterically regulating enzyme activities

Answer 8

s

Question 9

Relation of allosteric ligands to metabolic conditions

Answer 9

s

Question 10

Four common post-translational modifications of proteins, their chemical structure, process of their formation, functional outcome

Answer 10

w

Question 11

Membrane permeability

Answer 11

w

Question 12

Effects of osmotic pressure and water transport

Answer 12

s

Question 13

Protein pores

Answer 13

s

Question 14

Protein channels

Answer 14

s

Question 15

Protein transporters

Answer 15

s

Question 16

SGLT and changes in ligand transport and osmotic pressure

Answer 16

s

Question 17

Curve with inhibitory allostery?

Answer 17

1. Bind only DNA, tight Kd

2. Add binding of sugar; shift in Kd right, weaker binding

Question 18

Curve with enhancing allostery?

Answer 18

1. No purine binds at 10^-7 M

2. Add purine binding, shift curve to the LEFT; enhancing DNA binding

Question 19

Weighted average curve

Answer 19

In cooperativity, the average of a site with low affinity with that of a site with high affinity

High affinity = Takes less substrate to bind (more left on graph)

Question 20

Allosteric ligands as drugs (traditional and new possibilities)

Answer 20

Traditional: Drug competes with natural ligand in same binding site, inhibiting activity or substitutes for missing ligand

Newer: Work with drug at a different binding site on protein

1. More subtle effects: slow function, or ramp it up
2. Expands number of search targets (all proteins may have potential allosteric sites)

Question 21

Positive cooperativity (hallmark, graph, and nature)

Answer 21

1st event makes second stronger
Not much binding occurring, but once something does, 2nd event will be much more tight.

Hallmark: SLOPE is GREATER than 1

Graph: even on a linear scale, still shows sigmoidal characteristics at v low [ ].

Response range to to ligand change narrows
Dramatic biological effect
More like an on/off switch- once the protein is there, it can bind a lot more of it

Question 22

Negative cooperativity (hallmark, graph, and nature)

Answer 22

Binding one ligand makes subsequent binding more difficult

Hallmark: Never reaches 100% bound

Graph: Hyperbola is steeper at low concentrations, SLOPE is LESS than 1

Changes at higher ligand concentrations do not affect protein function: doesn’t have big fluctuations, but slow, continual responses over a wide range of concentrations

Constant protein function across a wide range of ligand concentrations

Question 23

Four ways that GLUT isoforms differ:

Answer 23

1. Substrate specificity
2. Substrate Km
3. Tissue distribution
4. Regulation

Question 24

Role of GLUT2. Why GLUT2 and not something with a stronger affinity?

Answer 24

GLUT2’s role is to move glucose from cell to blood
Runs completely on glucose gradient
Passive, facilitated diffusion requires sugar gradietn
Bi-directional transport

Good for its role because it never runs backwards.
Ex. In a fasting state, you don’t want glucose running back into cell
With a high Km value, there won’t be as much activity when patient is in the fasting state.