Regulation by Reversible Covalent

Modification

• For many enzymes, activity also is regulated by

reversible covalent modification of one or more amino
acid residues in the enzyme.
• Over 500 different types of covalent modifications
have been found in proteins. Common modifications
include phosphorylation, adenylylation, methylation,
myristoylation, and ubiquitination.
• In the case of ubiquitination, the small protein
ubiquitin is attached to a lysine residue in the target
protein flagging it for proteolytic destruction.
• The modifying groups are usually attached to a
regulated enzyme by a separate modifying enzyme
(e.g., a methylase in the case of methylation).
• Introduction of the modifying group alters the local
properties of the enzyme which typically undergoes a
change in conformation and activity.
• Introduction of a hydrophobic myristoyl group
triggers the association of the protein with a
membrane.
• Phosphorylation is the most important type of covalent
modification.
 Regulation by Reversible Phosphorylation

• It is estimated that one-third of all proteins in a eukaryotic cell are

phosphorylated, and one, or often many phosphorylation events are
part of virtually every regulatory process.
• Some proteins have only one phosphorylation site, whereas others
have several, and a few have dozens of sites for phosphorylation.
• The attachment of phosphoryl groups to specific amino acid residues
of a protein is catalyzed by enzymes called protein kinases.
• In these reactions, typically the -phosphoryl group from a nucleoside
triphosphate (usually ATP) is transferred to a particular Ser, Thr,
Tyr, or occasionally His residue in the target protein.
• Removal of the same phosphoryl group from the protein is performed
by enzymes called protein phosphatases.
• The introduced phosphoryl group can cause major changes in the
conformation of the modified enzyme by virtue of steric effects, or
hydrogen bonding and ionic interactions to neighboring residues.
• This can lead to effects on substrate binding and catalysis.
Regulation by Reversible Phosphorylation: Muscle Glycogen Phosphorylase

• An important example of enzyme regulation by phosphorylation is seen

with glycogen phosphorylase of muscle and liver.
• This enzyme catalyzes the breakdown of glycogen stores via the
reaction
• Glycogen(n) + Pi  glycogen(n-1) + glucose 1-P
• Glycogen phosphorylase is a homodimeric enzyme that exists in two
forms--the less active phosphorylase b form and the more active
phosphorylase a species (Fig. 6-36).
• Activation by phosphorylation is catalyzed by the enzyme phosphorylase
kinase which attaches phosphate groups to serine residues (Ser 14)
located near the N-termini of both subunits.

• The enzyme phosphoprotein

phosphatase 1 (PP1) removes these
two phosphates by hydrolysis,
inactivating the enzyme.
• The phosphorylation of Ser14 causes
major structural changes near the N-
termini of the glycogen phosphorylase
chains that produce a more active
enzyme.
• Note that glycogen phosphorylase is
also regulated by noncovalent
allosteric modulation
 Conformational Changes in Allosteric
Regulatory Enzymes
The modulators of an allosteric enzyme may be inhibitory or stimulatory.
If the modulator and substrate are the same the regulation is called homotropic.
If the modulator and substrate are different molecules the regulation is called
heterotropic.
Allosteric enzymes generally have one or more regulatory, or allosteric, sites for
binding the modulator.
Just as an enzyme’s active site is specific for its substrate, each regulatory site
is specific for its modulator.
In many allosteric enzymes, the substrate-binding and modulator-binding sites(s)
are on different subunits. These are called the catalytic (C) and regulatory (R)
subunits.

In Fig. 6-32, binding of the positive

(stimulatory) modulator (M) to its specific
site on the regulatory subunit is
communicated to the catalytic subunit
through a conformational change. This
change renders the catalytic subunit active
and capable of binding the substrate (S)
with higher affinity. On dissociation of the
modulator from the regulatory subunit, the
enzyme reverts to its inactive or less active
form
 Feedback Inhibition
• When a metabolic pathway is switched off by
its end-product.
• End-product usually inhibits an enzyme earlier
in the pathway.
• Prevents the cell from wasting chemical
resources

13
14
15
Isozymes