Phosphorylases, Phosphatases
and Kinases
The enzymes that deal with
phosphate
Phosphate serves biochemical systems in a major
way. High energy phosphate compounds such as ATP,
PEP, 1,3 bisphosphoglycerate owe their energetic
properties to anhydride bonds between neighboring
phosphates or phosphate, oxygen or nitrogen atoms.
This tutorial will introduce you to enzymes that deal with
phosphate in a biosynthetic and regulatory capacity.
These enzymes specifically add or remove phosphate
groups.
Phosphorylase (fos-for-a-lace)
The major function of this unique enzyme is to add a phosphate to glycogen
and by doing so, forms glucose-1-PO4. The reaction is classified as a phosphorylysis
because of its similarity to a hydrolysis (click 1). The only difference is a phosphate,
not a water molecule is placed across the bond (click 1). Click 1 to continue.
Hydrolysis
H-O-H
O
CH2OH
CH2OH
O
O
OH
O
OH
OH
O
O
O
O
O
O
OH
OH
OH
OH
OH
O
OH
O
HO
OH
Phosphorylysis
O
O
OH
CH2OH
OH
O
HO-P-OH
O CH2OH
CH2OH
OH
CH2OH
O
CH2OH
CH2OH
O
O
OH
O
O-P-O
OH O
OH
O
HO
OH
Pyrophosphorylases
(pyro-fos-for-a-laces)
These enzymes use nucleotide triphosphates (CTP, UTP, ATP, etc) to
make products with a pyrophosphate linkage. P~P is split out during the reaction.
Recall, a pyrophosphorylase activates glucose by making UDP-glucose from
glucose-1-PO4 and UTP (click 1). Note two features of the reaction: (1) the
formation of a pyrophosphate group in the product (click 1), and (2) the splitting
out of a P~P (click 1). The P~P is cleaved by an ever-present pyrophosphatase
yielding energy to drive the reaction (click 1). Pyrophosphorylases only work
when a phosphate is already in position on the molecule (click 1). Click 1 to go on.
CH2OH
CH2OH
O
O
OH
HO
+ UTP
OPO3
OH
=
uracil
O O
+
OH
O-P~O-P-O-ribose
O
HO
OH O
O O
O-P~O-P-O
O O
H2O
pyrophosphatase
2 HPO4=
Phosphatase (fos-fa-tace)
Phosphatases are hydrolases. This means these enzymes use a water molecule
to remove a phosphate group from a substate. As an example, observe the action of
glucose-6-phosphatase, a major enzyme that controls blood sugar. The substrate for
enzyme is glucose-6-PO4 (click 1). A water molecule is used to displace the phosphate
group from the molecule (click 1). Phosphatase reactions are NOT reversible. As a
guide to avoid confusion, remember that a phosphatase “takes off ”.
CH2OPO3=
O
OH
HO
OH
OH
CH2OH
+ H2O
X
O
OH
HO
+ HPO4=
OH
OH
You should also recall that a special class of phosphatases remove phosphate groups
from proteins. These so called “phosphoprotein phosphatases” control the activity of a
large class of enzymes that require phosphate groups for activity. Click 1 to go on.
Kinases (ki-naces)
Kinases are enzymes that typically transfer the terminal phosphate group
of ATP to an -OH group on a substrate (click 1). This results in a phosphate-ester
bond in the product. The reaction is not reversible. Although ATP is the major
substrate (click 1), occasionally GTP will be a phosphate group donor. Phosphate
groups in ATP are coordinated with a Mg2+ ion which strains the linkage between
the gamma and beta phosphate and facilitates the breakage of this bond (click 1).
The reaction is favored by the release of free energy that accompanies bond
breakage. Click 1 to go on.
R-OH + ATP
X
R-OPO3= + ADP
O O
-P-O-P-O-CH2 Adenine
O
O O
Mg2+
ATP
HO
HO
Summary of what have you learned?
Enzymes that transfer phosphate groups to substrates, relying
primarily on ATP as the phosphate donor, are call “kinases”.
A special type of enzyme has the capacity to add inorganic
phosphate groups across the glycosidic bonds of glycogen. This
enzyme is called “phosphorylase”. A phosphorylase can literally
cleave the glycosidic bond with a molecule of phosphate.
Enzymes that use nucleotide triphosphates to transfer a nucleotide
to a molecule, splitting out a pyrophosphate group in the transfer
are call “pyrophosphorylases”.
Enzymes that remove phosphate from the molecule are called
“phosphatases”.
(click 1 to go on)
Test you understanding
1. Name a kinase enzyme in the gluconeogenic pathway that requires
GTP as a phosphate donor?
PEP carboxykinase.
2. Why must the substrate for a pyrophosphorylase have a phosphate
group on the molecule?
In order to attach the nucleotide, the enzyme must make a pyrophosphate
linkage with the substrate. This requires a phosphate to be present.
3. To add a phosphate group to an unbound glucose molecule, you need
ATP. To add a phosphate to a glucose molecule that is part of glycogen,
you need only inorganic phosphate. Why?
Breaking the glycosidic bond provides the energy to drive the phosphorylation.
Free glucose has no glycosidic bond
4. Why does a cell require one enzyme to add a phosphate group and
another to take it off. Why 2 enzymes?
To reverse a kinase, ATP must be synthesized. Phosphate ester bond energy
does not supply sufficient energy.