PST 31215 Biochemistry II

Dr. (Mrs.) H. Sasimali M. Soysa

Senior Lecturer
Department of Physical Sciences and Technology
Faculty of Applied Sciences,
Sabaragamuwa University of Sri Lanka
1
Photosynthesis:Calvin Cycle
 Light reactions of Photosynthesis
generate ATP and NADPH

At thylakoid
membrane
in plants

• The ATP and NADPH are used to fix

carbon dioxide in the dark
reactions.
 The Calvin Cycle fixes CO2 into sugars

• In plant, long term energy storage in the form of sugars is produced by subsequent
sequence of light-independent reactions
 Rubisco catalyzes the
carboxylation of RBP

• CO2 is condensed with ribulose-1,5-biphosphate

to form an unstable intermediate that
hydrolyzes to two 3-Phosphoglycerates
• Catalyzed by ribulose 1,5-bisophosphate
carboxylase/oxygenase (Rubisco).
• Highly exergonic: G0’ = -51.9 kJ mol-1.
• Rate limiting step in hexose synthesis.
 RUBISCO
• Rubisco is the most abundant enzyme on
earth.
– Found on the stromal surface of thylakoid
membranes in chloroplasts.
– Has 8 large subunits (55 kDa, L) & 8 small
subunits (13 kDa, S).
– Each L chain has a catalytic site and a
regulatory site.
– Slow: maximal rate 3 s-1.
– A second CO2 must be bound to a lysine for
the enzyme to be active.
Rubisco Structure

CO2 is bound to
bind critical Mg2+
Rubisco Carboxylation on Mg2+
Rubisco also catalyzes oxidation of
Ribulose-1,5-bisphosphate

• Oxidation occurs at 25% the rate of

carboxylation at normal atmospheric conditions.
– 10 M CO2, 250 M O2
• Wastes Ribulose without fixing carbon.
• Requires the CO2 bound to lysine, so is
prevented when CO2 is low.
• Oxidation increases with temperature.
Recovery of Glycolate carbon
• 3 out of 4 carbons
are recovered from
2 glycolate
molecules via
conversion to Gly
and Ser.
• Process is called
photorespiration,
since O2 is used
and CO2 released.
 Production of hexose phosphates
• Process of Fructose and
Glucose phosphate generation
from 3-phosphoglycerate is
similar to Gluconeogenesis.
– GAP dehydrogenase uses
NADPH instead of NADH in
chloroplast.
– 3-phosphoglycerate can be
moved to the cytoplasm and
used for gluconeogenesis.
– Glc-1-P, Glc-6-P, and Frc-6-P
are interconvertable.
 The third phase is to regenerate
Ribulose-1,5-Bisphosphate.
• Use ketolase
and aldolase to
convert 3 and
6 carbon
sugars to a 5
carbon sugar.
• Convert to
Ribose and
Xylulose 5-
phosphates first.
Ribulose-1,5-Phosphate Production

What is wrong with the

sugar structure on this
slide?

• Ribose 5-P and Xylulose 5-P are readily

isomerized and epimerized Ribulose-5-P.
• Ribulose-5-P is phosphorylated to R-1,5-
Summary of Calvin Cycle

To make one hexose:

Sugars are stored as Starch & Sucrose
• Starch is built up from ADP-Glucose, similar to Glycogen
from UDP-Glucose
• Sucrose-6-P is made from Fructose-6-P and UDP-
Glucose, then dephosphorylated.
Pentose Phosphate Pathway

Synthesis of 5 Carbon Sugars and

NADPH in nonphotosynthetic
organisms.
 Requirements for NADPH
• Biosynthetic pathways & Detoxification require
NADPH as the reducing agent.
• Pathways requiring NADPH:
– Fatty acid biosynthesis
– Cholesterol biosynthesis
– Neurotransmistter biosynthesis
– Nucleotide biosynthesis
– Reduction of oxidized glutathione (detoxification)
– Cytochrome P450 monooxygenases
(detoxification)
 Tissues with Active Pentose
Phosphate Pathway
Tissue Function

Adrenal gland Steroid synthesis

Liver Fatty acid and cholesterol synthesis

Testes Steroid synthesis

Adipose tissue Fatty acid synthesis

Ovary Steroid synthesis

Mammary gland Fatty acid synthesis

Red blood cells Maintenance of reduced glutathione

Berg, Tymoczko, Stryer, 5th Ed., Table 20.4

Copyright © 2002, W. H. Freeman and Company
 Phases of Pentose Phosphate
Pathway
• The first phase of the
PPP is oxidative
decarboxylation to
generate NADPH.
• Gives 5-carbon sugars
for nucleotide
synthesis.
• Sugars are rearranged
in Phase 2.
 Oxidative Phase of PPP

• Two molecules of NADP are reduced in 3 steps.

• The ribulose-5-P can then be isomerized to
ribose-5-P by phophopentose isomerase.
Stage 2: Rearrangement of sugars
• Transaldolase and transketolase rearrange
sugars to link the PPP to glycolysis.
– Rearrangements similar to the Calvin Cycle.
• Reactions are reversible, so sugars can be
moved between the two pathways.
– If the main purpose is to make NADPH, the reactions
can be summarized as:
Net reaction:
 PPP rearrangement reactions

Rxn 1

Rxn 2

Rxn 3
 Effect of PPP Second Stage

Or, including conversion of ribose-5-P:

• Excess ribose-5-P produced in generating

NADPH can be converted to glycolysis or
glycolytic pathway intermediates.
• Ribose from diet can be used in glycolysis or
gluconeogenesis.
• Ribose-5-P can be generated for nucleotide
synthesis.
Four possible modes of using PPP
 Importance of NADPH

1. Required for the biosynthesis of fatty acids and

steroids
2. Free radical scavenging
3. Erythrocyte membrane integrity
4. Prevention of Met-Hemoglobinemia
5. Detoxification of the drugs (in liver)
6. Lens of eye
7. Macrophages bacterial activity
8. Availability of ribose
 Glutathione is reduced by NADPH

• Glutathione maintains a reducing environment in the

cytoplasm & protects against reactive oxygen species
(ROS).
• Glutathione reductase uses NADPH to reduce oxidized
glutathione (GSSG) to the reduced form (GSH).
• NADPH is generated by Glucose-6-P-dehydrogenase
(G6PD) & 6-Phosphoglucanate dehydrogenase of the PPP &
malic enzyme in the cytosol/mitochondrial acetylCoA
shuttle.
• RBC’s that have no mitochondria are susceptible to oxidative
damage in the case of G6PD deficiency.
– This is common in Thailand and other areas where malaria has
been prevalent.
– The oxidizing environment & lack of PPP products in the RBC
leads to poor growth of Plasmodium species.
 Summary
• The Calvin cycle uses NADPH and ATP to fix CO2 into
six carbon sugars, while the pentose phosphate pathway
(PPP) uses release of CO2 from a six carbon sugar to
generate NADPH.
• The Calvin cycle rearranges 6 and 3 carbon sugars to
regenerate the 5-carbon starting material (ribulose-5-
bisphosphate → ribulose-1,5-bisphosphate), while the
PPP rearranges the 5 carbon product to 3 and 6 carbon
sugars for use in glycolysis and glucose storage.
• Most steps are reversible, so the same enzymes can be
used in either direction for many of the PPP and Calvin
cycle steps.
• The directions and products of the pathways are
detemined by cellular needs.
Glycogen & Starch
metabolism
Glucose storage
 Glycogen metabolism
• Glycogen is a common and efficient storage
form of glucose
• Glycogen is synthesized and broken down by
different pathways.
– (glycogenesis) Synthesized by Glc-6-P > Glc-1-
P>UDP-Glc > Glycogen
– (glycogenolysis) Broken down by phosphorylase:
Glycogen + Pi > Glc-1-P > Glc-6-P
• Glycogen synthesis and degradation are
reciprocally regulated.
– Synthesize when fuel levels are high.
– Breakdown when Glucose (liver/blood) or energy
(muscles) is needed.
 Glycogen Breakdown

• Glycogen 1,4-Glc are removed

by
glycogen phosphorylase.
• Glycogen is debranched near
1,6 branchpoint by moving 3
glucose from branch to end.
• Alpha-glucosidase removes
the final glucose from the
branch.
• Phosphorylase continues.

Phosphorylase rxn Alpha-1,6-glucosidase rxn

 Glycogen Synthesis
• Glycogen starts from a
glycogenin dimer, which
synthesizes an -1,4-
linked glucose chain
linked to a tyrosine.
• Glycogen synthase
adds more Glc to this
chain from UDP-Glc.
• A branching enzyme
transfers 7 Glc
residues from a chain
at least 11 Glc long to
make each branch.

Glycogen synthase
 Glycogen metabolism regulation
Either
epinephrine
(in muscles)
or glucagon
(in liver)
can
stimulate
adenylate
cyclase.

• Hormone stimulated phosphorylation of phosphorylase

activates it, increasing glycogen breakdown.
• Hormone stimulated phosphorylation of Glycogen
synthase inactivates it, decreasing glycogen synthesis.
• Phosphatase I can take these phosphates off, reversing
the effects. It is stimulated by insulin.
Starch synthesis occurs in plastids
• Starch is synthesized from ADP-Glc
– In chloroplasts, Glc is made from triose phosphates
produced in the Calvin cycle.
– 3-phosphoglycerate phosphorylated to 1,3-
bisphosphoglycerate with ATP
– 1,3-bisphosphoglycerate reduced to Glyceraldehyde-
3-P (GAP) with NADPH.
– Triose phosphate isomerase converts half of GAP to
Dihydroxyacetone phosphate (DHAP)
– Aldolase converts GAP + DHAP to Fructose-1,6-
bisphosphate (F1,6BP)
– F1,6BP is dephosphorylated to Fructose-6-P >
Glc6P> Glc1P.
 Overall picture of starch synthetic pathway

GAP DHAP

aldolase
Fructose 1,6-P

Fructose 1,6-bisphosphatase

Fructose 6-P
glucose 6-phosphate isomerase

Glucose 6-P
phosphoglucomutase

Glucose 1-P
ADP-glucose
AT pyrophosphorylase
P
ADP-
Glucose

Glc n

Glcn+1

GAP = glyceraldehyde-3-P, DHAP = dihydroxyacetone P

Starch synthesis in amyloplast
• Amyloplast is white
plastid for starch
synthesis & storage.
• Have ADP-Glc, Glc
and Glc-6-P
transporters.
– Glc-6-P transporter
exchanges Glc-6-P
for Pi.
– ADP-Glc transporter
exchanges ADP-Glc for
AMP
– Hexokinase
phosphorylates Glc
after transport into the
amyloplast.
 Synthesis of Starch
• Three enzymes for starch synthesis:
– ADP-glucose pyrophosphorylase
– Starch synthase
• Different isoenzymes elongate amylose chains on amylose and
amylopectin.
– Soluble isoenzymes add -1,4-linked glucose to amylopectin
– Granule-bound isoenzymes (mainly waxy) add -1,4-linked
glucose to
amylose inside starch granules.
– Starch-branching enzyme
• Moves -1,4-linked chain to a 6-OH approx. 20 Glc residues from
the nonreducing end to make an -1,6-branch.
• Two forms, I & II
– Form I adds to amylose & less branched amylopectin
– Form II adds more shorter branches to highly branched
amylopectin.
• Different starches have different distances between
branches and proportions of amylose to amylopectin.
Synthesis of amylose and amylopectin
 Summary of Glycogen & Starch
Synthesis & Breakdown
• Glycogen and starch synthesis both involve synthesis of
-(1,4)-glucosyl chains and transferring to make -(1,6)-
linked branches.
• Glycogen synthesis uses UDP-Glc as the precursor,
while starch synthesis used ADP-Glc.
• Phosphorylase breaks down glycogen to Glc-1-P with
help from debranching enzyme and -glucosidase.
– Alpha-glucosidase produces glucose, rather than glucose-1-
P
– Breakdown of starch can be similar or be done by hydrolytic
amylases.
• Synthesis & breakdown must be carefully controlled to
make sure only one happens at one time.