Biochem Mechanisms for Regulating Metabolism (LSK) Flashcards
Protein-ligand binding
The affinities for each successive ligand is different, but the pathways from start to finish are energetically equal
Cooperativity: features, examples, and binding curve (+, -)
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
Allostery: features, examples in nature, and binding curve
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
Function, tissue distribution, and regulation of GLUT isoforms
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Km values to physiological concentrations of ligand
Km = concentration of substrate that allows enzyme to be at half of maximal velocity
Kd values to physiological concentrations of ligand
Kd = the concentration at which 50% of binding sites (receptors) are occupied by the drug
Mechanisms for altering substrate/metabolite concentration and consequence on enzyme activity
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Strategies for allosterically regulating enzyme activities
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Relation of allosteric ligands to metabolic conditions
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Four common post-translational modifications of proteins, their chemical structure, process of their formation, functional outcome
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Membrane permeability
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Effects of osmotic pressure and water transport
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Protein pores
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Protein channels
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Protein transporters
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SGLT and changes in ligand transport and osmotic pressure
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Curve with inhibitory allostery?
- Bind only DNA, tight Kd
2. Add binding of sugar; shift in Kd right, weaker binding
Curve with enhancing allostery?
- No purine binds at 10^-7 M
2. Add purine binding, shift curve to the LEFT; enhancing DNA binding
Weighted average curve
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)
Allosteric ligands as drugs (traditional and new possibilities)
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
- More subtle effects: slow function, or ramp it up
- Expands number of search targets (all proteins may have potential allosteric sites)
Positive cooperativity (hallmark, graph, and nature)
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
Negative cooperativity (hallmark, graph, and nature)
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
Four ways that GLUT isoforms differ:
- Substrate specificity
- Substrate Km
- Tissue distribution
- Regulation
Role of GLUT2. Why GLUT2 and not something with a stronger affinity?
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.
