SOLOMON, Marc Ralph M.

2015-48305 Biochem 34
WORKSHEET 2

GLUCOSE 6-PHOSPHATASE
Glucose 6-phophatase (G6Pase), which is mainly found in the liver, is the enzyme that is responsible
for gluconeogenesis and glycogenolysis. Gluconeogenesis is a metabolic pathway that results in the
generation of glucose from non-carbohydrate carbon substrates such as lactate, glycerol, and
glucogenic amino acids. Lactate in the Cori cycle is produced by anaerobic glycolysis in the muscle
which is moved to the liver where the G6Pase will convert it to glucose. For glycogenolysis, glycogen
stored in the liver and muscles are converted to glucose 6-phosphate which is hydrolyzed to produce
glucose in the liver through G6Pase. For these reasons, G6Pase is only found in the liver and not in
myocytes or muscle cells (Shaftingen & Gerin, 2002; Nelson & Cox, 2008).

1. How many polypeptide chains make up your enzyme? Can you describe the primary,
secondary, supersecondary, tertiary and quaternary structure of your enzyme? Is your
enzyme part of supramolecular structures? Explain.

Mammalian G6Pase is composed of 357 amino acids and according to Pan, et al., 1998,
G6Pase complex is also comprised of four components, the glucose-6-phosphate catalytic
subunit (G6PC1), the glucose-6-phosphate transporter (T1), inorganic phosphate transporter
(T2), and glucose transporter (T3), all of which are ER-membrane bound proteins. However,
due to its intact association to the endoplasmic reticulum, the present knowledge about its
exact structure is still limited (Ghosh, et al., 2002).

2. Show the mechanism of the reaction catalyzed by your enzyme. Include the role of the
cofactor/ coenzyme in the reaction.
(Ghosh, et al., 2002)
3. For the enzyme with kinetic data shown below, plot the Lineweaver-Burke line and determine
Km, and Vmax of the uninhibited enzyme. What kind of inhibition is observed? Explain.
[S] M V (mol/min) with V (mol/min) with V (mol/min) with
0.0 nM Inhibitor 25 nM Inhibitor 50 nM Inhibitor
0.4 0.22 0.21 0.20
0.67 0.29 0.26 0.24
1.00 0.32 0.30 0.28
2.00 0.40 0.36 0.32

a. y = 0.9997x + 2.032
Vmax=0.49mol/min
KM=0.488 mol/min
b. y = 0.9865x + 2.3252
Vmax= 0.43mol/min
KM= 0.185mol/min
c. y = 0.9474x + 2.6649
Vmax= 0.38mol/min
KM= 0.36mol/min

Since the both Vmax and KM decreased upon addition of the inhibitor, the kind the of inhibition is
uncompetitive.

4. How is the activity of your enzyme regulated?

The physiological activity of G6Pase is regulated by substrate concentration. It is further

activated when glycogen degradation and/or gluconeogenesis are stimulated by adrenergic
hormones or by glucagon which is secreted by the cells of pancreas when the blood-sugar level
is low. Glucagon activates the glycogen metabolism in hepatocytes by inhibiting pyruvate kinase
which catalyzes the final step of glycolysis. On the other hand, when G6Pase is excessively
active, the control of its catalytic activity depends on the inhibitors such as glucose which is a
non-competitive inhibitor. Another well-known inhibitor of phosphatases is vanadate which forms
phosphoenzyme-intermediate which inhibits the phosphohydrolase and phosphotransferase
activities of G6Pase (Schaftingen & Gerin, 2002).

5. A novel enzyme was detected in the liver of a sea animal. Describe the steps you will
undertake to isolate and characterize the enzyme.

In order to isolate the protein detected from the liver of the sea animal, the liver cells should,
firstly, be lysed either by physical or chemical processes. Physical means include the use of
hypotonic solution to induce bursting of the cell, homogenization, and sonification. Whereas for
the chemical methods, they include using of detergents, like those with fatty and polar molecules,
or enzymes, like proteases and lipases. In the case of liver cells, homogenization is enough to
 lyse the cells which is done usually at 4C to prevent denaturation. The next step will be
fractionation which can be done either through salting out using NH4OH, which unfolds and
precipitates out the enzyme, or through differential centrifugation that uses centrifugal and
gravitational force to create fractions. Next will be the purification of the obtained protein solution
which involves the removal of other proteins aside from the protein of interest. There are several
processes to purify the protein solution such chromatography that includes size exclusion, ion
exchange, reverse-phase, and affinity chromatography. For the purification of the enzyme from
the liver of the sea animal, affinity chromatography is enough to get the target protein. This type
of chromatography has a stationary phase with specific ligand where the protein target will attach
and those nontarget proteins will be eluted out first.

6. Having purified your enzyme, what assay methods can be used to determine whether you
have indeed isolated your enzyme? What methods do you use to determine purity? Yield?

To determine whether the target enzyme is indeed isolated, isoelectric focusing followed by
SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Cell Electrophoresis) can be done. Using
these methods, you will know if you successfully isolated the protein if its stain, based on its pI
and molecular weight, is present. Purity is also possible to be determined using these methods
because if you isolated enzyme is impure, several stains will appear. As for the yield, protein
quantitations are used like Biuret, Bradford, Lowry protein assays.

7. What methods can you use to determine the following? Describe these
a. Sequence of your enzyme

To determine the sequence of the enzyme, Edman degradation is usually used. This method
uses phenyl isothiocyanate which reacts with the N-terminus of the protein/enzyme. Upon
addition of acid, the amino acid at the N-terminus is detached from the polypeptide chain
and, thus, can be identified using NMR. The advantage of Edman degradation is that it leaves
the rest of the chain intact since there is no hydrolysis involved. After identifying the first
amino acid, the new N-terminus can now be again identified by doing another round of
reaction with phenyl isothiocyanate. However, this is only practical to use for short peptide
chains. For longer or large proteins, unfolding is first done by breaking disulfide bonds using
-mercaptoethanol, and then cleaving or fragmenting the polypeptide chain which can be
done using cyanogen bromide which cleaves at the carbonyl side of methionine residues.
Other way to cleave the chain is using proteases such as trypsin, chymotrypsin, and
staphylococcus. Trypsin cleaves at the carbonyl side of lysine and arginine residues;
chymotrypsin cleaves at carbonyl side of aromatic residues such as tyrosine, tryptophan, and
phenylalanine; and staphylococcus cleaves at the aromatic side of the aspartate and
glutamate.

b. N-terminal amino acid

N-terminus amino acid can be determined using Sanger method which involves the use of
2,4 dinitrofluorobenzene (DNFB) or other reagents such as dansyl chloride and dabsyl
chloride. DNFB reacts with the N-terminus of the enzyme detaching it from the chain which
makes it possible to subjected to NMR for identification. However, this method hydrolyses
the whole peptide chain which is why it is not used for sequencing of the whole chain. An
alternative method that can be used is the Edman degradation.
 c. Presence of methionine residues

The presence of methionine residues is usually determined using cyanogen bromide

reagent which hydrolyzes the peptide bond at the C-terminus of methionine.

d. Presence of basic residues

Precipitation method using silver (Ag+) indicates presence of basic residues. Moreover,
other enzymes can also be used like trypsin which cleaves at polypeptides at their basic
residues.

e. Presence of acidic residues

Using staphylococcus, peptide chains are cleaved at the carbonyl side of aspartate and
glutamate which are acidic amino acid residues. Aside from this, hydrogen peroxide is also
used which reacts with acidic residues to form carboxylic acid, ammonia, and carbon
dioxide.

Ghosh, A., et al. (2002). The catalytic center of glucose 6-phosphatase. The Journal of Biological
Chemistry, 277(36), 32837-32842. doi: 10.1074/jbc.M201853200.

Nelson, D. L. & Cox, M. M. (2008). Lehninger: Principles of biochemistry, 5th Ed. New York: W. H.
Freeman and Company.

Pan, C. J., et al. (1998). Transmembrane topology of glucose 6-phosphatase. The Journal of
Biological Chemistry, 273(11), 6144-6184. doi: 10.1074/jbc.273.11.6144.
Schaftingen, E. V. & Gerin, I. (2002). The glucose 6-phophatase system. Biochemical Journal, 362,
513-532. Retrieved 19 September 2017 from https://www.ncbi.nlm.nih.gov/pubmed/11879177