Reversible covalent modification of phosphorylase enzyme

1. REVERSIBLE COVALENT
MODIFICATION OF
PHOSPHORYLASE
HEMA T
MSc BIOCHEMISTRY
BHARATHIAR UNIVERSITY
2018-2020

2. PHOSPHORYLASE
 Phosphorylases are enzymes that catalyze the addition of a phosphate
group from an inorganic phosphate (phosphate + hydrogen) to an
acceptor.
A-B + P ⇌ A + P-B
 Enzyme class : Transferase
 EC Number : 2.4 and 2.7.7

3. CLASSES OF PHOSPHORYLASE
 Glycosyltransferases (EC 2.4)
Enzymes that break down glucans by removing a glucose residue (break O-glycosidic
bond)
glycogen phosphorylase
starch phosphorylase
maltodextrin phosphorylase
Enzymes that break down nucleosides into their constituent bases and sugars
(break N-glycosidic bond)
Purine nucleoside phosphorylase (PNPase)

4.  Nucleotidyltransferases (EC 2.7.7)
Enzymes that have phosphorolytic 3' to 5' exoribonuclease activity
phosphodiester bond)
RNase PH
Polynucleotide Phosphorylase (PNPase)

5. REGULATORY ENZYMES
 Each metabolic pathway includes one or more enzymes that have a
greater effect on the overall sequence are called regulatory enzymes.
 In most multienzyme systems, the first enzyme of a system is a
regulatory enzyme.
 The activity of regulatory enzymes are modulated in a variety of ways.

6.  Allosteric enzymes function through reversible, non covalent binding of
regulatory compounds called allosteric modulators or allosteric effectors
which are generally small metabolites or cofactors.
 Other enzymes are regulated by reversible covalent modification.

7. REVERSIBLE COVALENT MODIFICATION
 Important class of regulatory enzymes, activity is modulated by covalent
modification of one or more of the amino acid residues in the enzyme
molecule.
 Over 500 different types of covalent modifications have been found in
proteins.
 Common modifying groups include phosphoryl, acetyl, adenylyl, methyl,
amide, carboxyl, myristoyl, palmitoyl, prenyl, hydroxyl, sulphate and
ADP- ribosyl groups.
 There are even entire proteins that are used as entire modifying groups,
including ubiquitin and sumo.

8. GLYCOGEN PHOSPHORYLASE
 An important example of enzyme regulation by reversible covalent
modification of enzyme is seen in glycogen phosphorylase of muscle
and liver (phosphorylation and dephosphorylation).
(Glucose)n + Pi (glucose)n-1 + glucose 1- phosphate
glycogen shortened
glycogen
chain

9. GLYCOGEN PHOSPHORYLASE OCCURS IN
TWO FORMS
 Phosphorylase a
― phosphorylated
― more active
― default liver enzyme
 Phosphorylase a has two subunits each with a specific serine residue
that is phosphorylated at its hydroxyl group.
 These serine phosphate residues are required for maximal activity of the
enzyme.
The phosphoryl groups can be hydrolytically removed by a separate
enzyme called phosphorylase phosphatase.

10. Phosphorylase a + 2H2O Phosphorylase b + 2Pi
(more active) (less active)
Phosphorylase a is converted to phosphorylase b by the cleavage of two
serine phosphate covalent bond one on each subunit of glycogen
phosphate.

12. PHOSPHORYLASE b
 Dephosphorylated
 Less active
 Default muscle enzyme
Phosphorylase b can in turn be reactivated- covalently transformed back
into active phosphorylase a by another enzyme, phosphorylase kinase,
which catalyses the transfer of phosphoryl groups from ATP to the hydroxyl
groups of the two specific serine residues in phosphorylase b.

13. 2ATP + Phosphorylase b 2ADP + Phosphorylase a
(less active) (more active)

15. REGULATION OF GLYCOGEN
PHOSPHORYLASE
The breakdown of glycogen in skeletal muscles and the liver is
regulated by variations in the ratio of the two forms of
glycogen phosphorylase.
The regulation of glycogen phosphorylase by phosphorylation
illustrates the effects on both structure and catalytic activity of
adding a phosphoryl group.

17. Phosphorylase b Phosphorylase a

18.  In the unphosphorylated state, each subunit of this enzyme is folded so as to bring the
20 residues at its amino terminus, including a number of basic residues, into a region
containing several acidic amino acids; this produces an electrostatic interaction that
stabilizes the conformation.
 Phosphorylation of Ser14 interferes with this interaction, forcing the amino-terminal
domain out of the acidic environment and into a conformation that allows interaction
between the P -Ser and several Arg side chains. In this conformation, the enzyme is much
more active.
 Phosphorylation of an enzyme can affect catalysis in another way: by altering substrate-
binding affinity. For example, when isocitrate dehydrogenase (an enzyme of the citric
acid cycle) is phosphorylated, electrostatic repulsion by the phosphoryl group inhibits the
binding of citrate (a tricarboxylic acid) at the active site.

19. THANK YOUTHANK YOU