An investigator has a purified sample of muscle phosphorylase b that is known to

Question

An investigator has a purified sample of muscle phosphorylase b that is known to be relatively inactive. a) Suggest two different methods that she could use to activate the enzyme. b) Once she has activated the enzyme she sets up a reaction containing (ONLY!) the following components: 25 uL of a stock solution of unbranched glycogen, 3.00 mL of 0.1 M HEPES buffer, pH 7.5, 25 uL of a 100 uM stock solution of GTP. She is surprised to see NO activity in her assay when she adds a small amount of enzyme. Explain to her what she did wrong and suggest a solution to her problem

Explanation / Answer

a) The enzyme can be activated by:

i) activating the cAMP-regulated G protein receptor system. This will cause the production of enzyme kinase that will phosphorylate the muscle enzyme. Similar kind of activation would result by activating the Ca2+ regulated G protein receptor system.

ii) increasing the AMP levels in the muscle to provide allosteric regulation.

b) The enzyme needs a cofactor called pyridoxal phosphate (PLP) that is important in carrying out the reaction. Also, since the enzyme is already activated, an addition of GTP will only act as a negative regulator/ inhibitor.

So, rather than adding GTP, one can add molecules like AMP which are positive regulators of the enzyme.

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