Calvin Cycle & Pentose Phosphate Pathway

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

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: DG0′ = -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-1,6-bisphosphate Oxidation occurs at 25% the rate of carboxylation at normal atmospheric conditions. 10 mM CO2, 250 mM 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 in chloroplast uses NADPH instead of NADH. 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 transketolase and aldolase to convert 3 and 6 carbon sugars to a 5 carbon sugar. Convert to Ribose and Xylulose 5-phosphates first.

Ribulose-1,6-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-BP.

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 Neurotransmitter biosynthesis Nucleotide biosynthesis Reduction of oxidized glutathione (detoxification) Cytochrome P450 monooxygenases (detoxification)

Tissues with Active Pentose Phosphate Pathway Function Adrenal gland Steroid synthesis Liver Fatty acid and cholesterol synthesis Testes Adipose tissue Fatty acid synthesis Ovary Mammary gland 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: Transaldolase

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

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-phosphate → 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. Synthesized by Glc-6-P > Glc-1-P>UDP-Glc > Glycogen 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. Alpha-1,6-glucosidase rxn Phosphorylase rxn

Glycogen Synthesis Glycogen starts from a glycogenin dimer, which synthesizes an a-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 ATP ADP-glucose pyrophosphorylase Pi 3-PG ADP-Glucose Pi from exchange of triose phosphates with cytoplasm; 3-PG from photosynthesis. Glc n Glcn+1 Slide: Rodjana Opassiri 3-PG = 3-phosphoglycerase, 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 a-1,4-linked glucose to amylopectin Granule-bound isoenzymes (mainly waxy) add a-1,4-linked glucose to amylose inside starch granules. Starch-branching enzyme Moves a-1,4-linked chain to a 6-OH approx. 20 Glc residues from the nonreducing end to make an a-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 a-(1,4)-glucosyl chains and transferring to make a-(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 a-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.