March 11 Flashcards
What are the 3 mechanisms that the AMPK complex is activated (or kept activated) by AMP or ADP?
- Have promotion of phosphorylation of Thr-172 on activation loop by an upstream kinase, so now active and on
2.Allosterically activated by AMP (once phosphorylated first), increases activity by 2-10 fold - Inhibition of the dephsophorylation of the activation loop, done by AMP and ADP binding to CBS domains of the gamma subunit of AMPK
How did western blots give us the knowledge that AMP, ADP, ATP do or don’t protect AMPK from activation/deactivation?
We have antibody that will only bind to the phosphorylated (activated) AMPK. Control used to make sure total amount of the aphla subunit was constant. THen Find by adding AMP only, did fold activation increase of AMPK. Then see that AMP and to a lesser extent ADP both protect AMPK from dephosphorylation when phosphatase present, but ATP does not, and at a certain conc. it will outcompete ADP and AMP and allow AMPK to be dePO4’d.
What 3 reactions buffer the amount of AMP, ADP when ATP levels decrease? What is the ideal ratio of these substrates?
Have ADP and Pi —> ATP
THen The reverse of the above
THen have 2 ADP <–>ATP+AMP
Ideally have AMP:ADP:ATP
1:10:100
What enzyme will do 2 ADP <—> ATP + AMP reaction?
It is adenylate kinase
IF ADP:ATP ratio rises by factor of 5, how much does AMP:ATP ratio raise by?
As it is [AMP][ATP]/[ADP]^2, then it is 5^2 giving 25 increase of AMP:ATP ratio
Studys show that the AMP concentration must be quite high to compete with the total ATP concentration for binding to CBS domains. What is a different form of ATP, how affect binding and how much AMP needed?
Have Mg-ATP, it does not bind as good to CBS domains, and as the ratio of free ATP:MgATP is 1:20, then AMP is actually able to outcompete it at a much lower concentration than for free ATP
THe AMPK Y subunit has 4 sites (CBS domains) for binding AMP, ADP, ATP. What site numbers are fought over for binding, which is the most important and why for AMPK regulation?
Sites 1 and 3 seem to have AMP in competition with ADP and ATP. It seems that site 3 is the most important as the alpha hook has end at this site 3, and the other end of the hook connects to regulatory interface that can shift to block the active site if needed.
When AMP only is bound, AMPK is a better substrate for ____1____. When AMP or ADP are bound to CBS domains, AMPK is a worse substrate for ____2____. What is conclusion from this?
1 = kinase kinase
2= phosphatase
Conclude that only AMP binding will help AMPK get phosphorylated, but either AMP or ADP binding will help prevent it from being dephosphorylated
Why does mutation in gamma isoform 2 subunit of AMPK CBS domains cause hypertrophic cardiomyopathy? What big factors change in cells?
Have higher basal activation loop phosphorylation so higher AMPK activity. This means more GLUT4 expressed on plasma MB so more glucose import, and becauase too much of it, there is lots of glycogen produced. We also see from activated AMPK that ACC is phosphorylated and now deactive, so less malonyl-CoA produced, so less FA biosynthesis, but also have more breakdown of FA’s to CO2 and ATP through FA oxidation
How many isoforms of each of the AMPK subunits? What ist h
alpha=isoforms 1 or 2
beta=isoforms 1 or 2
gamma = isoforms 1,2,3
A drug that binds in hydrophobic cleft between Beta and alpha subunit of AMPK and allosterically activates it, is it specific for AMPK, or would targeting active site of AMPK be better? Could be used to treat what?
It is more selective than just targeting the active site as many active sites would be similar to AMPK (kinase active sites). The cleft between the subunits is unique to AMPK, so less side effects. Activating AMPK with drug on different part of the enzyme could be used to treat diabetes by enhancing glycogen storage and increased glucose uptake
Do long chain fatty acid CoA’s activate AMPK? Direct or indirect if they do?
They directly activate AMPK.
