Chapter 20 The Calvin cycle and Pentose Phosphate Pathway

an evolutionary kinship

§ Light reaction (Ch. 19) § Dark reaction (Calvin cycle)

Calvin cycle – take place in the stroma of chloroplast

6 CO2 + 18 ATP + 12 NADPH + 12 H2O C6H12O6 + 18 ADP + 18 Pi + 12 NADP+ + 6 H+

3 stages:

or 10

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step

G°= 12.4 kcal/mol

Tracing the fate of CO2

(C3 plant)

time course

Rubisco located on the stromal surface of the thylakoid membrane;

probably the most abundant protein in the biosphere;

8 large subunits: each contains catalytic site and regulatory site

8 small subunits: enhance the catalytic activity of L subunits

a slow enzyme (kcat), 3 s-1

requires a magnesium ion and a CO2 other than the substrate

CO2

the activity increase markedly on illumination (CAM?)

also catalyzes oxygenase reaction – photorespiration

Rubisco activase faciliate

How to mimic?

active form

substrate

Rubisco activase:

exhibit an ATPase activity

two polypeptide (42 and 47 kDa)

Taiz and Zeiger, 2006

Oxygenase activity of Rubisco

2C

still requires that Lys210 be in the carbamate form

no CO2, no oxygenation

The rate of carboxylation is four times that of oxygenation under normal

conditions.

Stromal [CO2]: 10 M, [O2]: 250 M

A salvage pathway for phosphoglycolate

Photorespiration a wasteful process

¾ C

TPP

23C

16C + 33C

35C

5C

6 CO2 + 18 ATP + 12 NADPH + 12 H2O

C6H12O6 + 18 ADP + 18 Pi + 12 NADP+ + 6 H+

53C

6(3CO2)

35C

+ 3 H2O

10

Rubisco activity analysis?

Synthesis of sucrose and startch in cytoplasm and chloroplasts, separately.

Phosphate translocator

Storage forms in plants: sucrose, starch, fructan

ADP-G starch

(diel)

§ 20.2 The activity of the Calvin cycle depends on

environmental conditions

alkaline pH and [Mg2+]

rhythm phenomenon

Thioredoxin: plays a key role in the regulating the Calvin cycle a 12 kDa protein that have a catalytically active disulfide group containing two redox-active cysteine residues (-Trp-Cys-Gly-Pro-Cys).

Calvin cycle

C4 pathway of tropical plants sugar cane

PEP carboxylase

Malate dehydrogenaseMalic enzyme

concentrating CO2 , little phosphorespiration

2 ATP

Pyruvate-Pi dikinase

6 CO2 + 30 ATP + 12 NADPH + 24 H2O C6H12O6 + 30 ADP + 30 Pi + 12 NADP+ + 18 H+

Kranz (wreath) cells:

Crassulacean acid metabolism (CAM)Cacti, pineapple, vanilla, agave and mature

Phalaenopsis. Spatial and temporal

Water use efficiency

Nocturanl acidification

§ 20.3 Pentose Phosphate Pathway in plants and

animal generate NADPH, CO2, and 5C sugar.

5C sugar is components of RNA, DNA, ATP, NADH, FAD, CoA.

take place in cytoplasm

+ CO2

G6P dehydrogenase

[gluconeogenesis]

35C26C + 13C (p. 579)

transaldolase

isomerase epimerase

Oxidative phase of PPP

Phase 1

Glucose 6-P + 2 NADP+ + H2O

ribose 5-P + 2 NADPH + 2 H+ + CO2

Intramolecular ester

C-1 carboxyl group

C-5 hydroxyl group

C-1

Nonoxidative phase of PPP

transketolase and transaldolase (Ex.1)

linked between PPP and glycolysis

(2C)

-Ketoglutarate dehydrogenase pyruvate dehydrogenase

TPP

Transaldolase DHAP

(3C)

Transketolase

Transketolase: 2C

Transaldolase: 3C

C-3 and C-4 is split

Carbanion intermediates formation – the common feature of transketolase and

transaldolase

The rate of PPP is controlled by the level of NADP+

3 (5C) 2 (6C) + 1 (3C)

§ 20.4 The fate of glucose 6-phosphate – the need for NADPH, ribose 5-P, and ATP

5C NADPH

e.g., rapidly dividing cells

Net: 5 G6P + ATP 6 R5P + ADP + H+

5C = NADPH

Net: G6P + 2 NADP+ + H2O R5P + 2 NADPH + 2H+ + CO2

5C NADPH, G6P CO2

Net: G6P + 12 NADP+ + 7 H2O 6CO2+ 12 NADPH + 12H+ + Pi

The cytoplasm of a live cell from a well-fed rat:

NADP+/NADPH 0.014≒

NAD+/NADH= 700

Both NADPH and ATP are required

Net: 3 G6P + 6 NADP+ + 5 NAD+ + 5 Pi + 8 ADP

5 pyruvate + 3 CO2+ 6 NADPH + 8H+ + 5 NADH + 2 H2O + 8 ATP

ATP

Calvin cycle:

CO2 fixation

to use NADPH

C6 + C3 C5 in regeneration phase

PPP:

CO2 production

to produce NADPH

C5 C6 + C3

Reactive oxygen species removed

superoxide dismutase (p. 518) catalase peroxidase (glutathione, ascorbate) G6P dehydrogenase reduced glutathione (GSH)GSSG reduced ascorbate G6P dehydrogenase level [NADPH] sensitive to oxidative stress

especially important in red blood cells

Phytochelatin

(-Glu-Cys)n-Gly

n= 2~7

The functions of reduced glutathione (GSH)

Serves as a sulfhydryl buffer

maintains the cysteine residues of hemoglobin and

other red-blood-cell proteins in the reduced state.

in normal red blood cells: [GSH]/[GSSG] 500

To maintain the normal structural of red blood cells

To keep hemoglobin in the ferrous state

To detoxify hydrogen peroxide and organic peroxides

glutathione peroxidase: 2 GSH + ROOH GSSG + H2O +

ROH

§ How to regenerate GSH

mediate glutathione reductase – a flavoprotein

NADPH FAD-Enz (FADH2-Enz)

Enz-cys-cys-Enz (Enz-cys-SH)

GSSG (GSH)

Glucose 6-phosphate dehydrogenase– plays a key role in protection against ROS

Glucose 6-phosphate dehydrogenase deficiency– is inherited on the X chromosome

– pamaquine, a purine glycoside of fava beans, leads to the generation of peroxides, then induced hemolytic anemia

– urine turned black, jaundice developed, and the hemoglobin content of the blood dropped sharply

– cross-linked hemoglobins to form Heinz bodies on cell membrane caused membrane damage and cell lysis

Glucose 6-phosphate dehydrogenase deficiency– not all bad

protect against falciparum malaria

the parasites required GSH and the products of PPP for optimal growth

11% among Americans of African heritage

the interplay of heredity and environment in the production of disease

atypical reactions to drugs may have a genetic basis

瘧疾分為四種，其中，最嚴重的是惡性瘧 （ falciparum malaria ），這種瘧疾會對生命造成威脅。 其他三種瘧疾 -- 隔日瘧、三日瘧卵圓形瘧疾、(vivax, malariae 及 ovale) 則較不嚴重，並且沒有立即致命的危險。

Fenton reactionfrom plant physiol biochem (2000) 38: 125-140

The exceedingly active oxygen species can be produced in a Fenton reaction involving Cu+ and H2O2

O2 ＋ AH2 → H2O2 ＋ A

 

2 Cu2+ ＋ AH2→ 2 Cu+ ＋ A ＋ 2 H+

 

Cu+ ＋ H2O2 → Cu2+ ＋ OH ＋ OH-

96T (191)

97T

95C

The reactions of pentose phosphate pathway operate exclusively in (A) mitochondria, (B) cytoplasm, (C) chloroplast, (D) ribosome, (E) endoplasmic reticulum.

97C

98T