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.

Answer 1

(a). Two different methods to activate the enzyme muscle phosphorylase b are -

(1). By adding AMP (adenosine monophosphate) to the sample.

(2). By using active phosphorylase kinase and ATP to phosphorylate the enzyme.

(b). Reaction of activated enzyme and the unbranched glycogen does not occur due to the absence of uncleaved glycosal units. To proceed the reaction Inorganic phosphate is also required for the conversion of glycosyl residues in glycogen to glucose 1-phosphate.