Advanced Biochemistry - Metabolism 45 questions assignment

Chapter 19 The Dark Reactions

Key Concepts 19.3 • The Calvin cycle carboxylates a pentose, converts the products to glyceraldehyde-3-phosphate, and regenerates the pentose, using the ATP and NADPH produced by the light reac@ons. • The products of the Calvin cycle are converted to carbohydrates (glucose polymers). • The Calvin cycle enzymes are more reac@ve in the light. • Plants undergo photorespira@on, which consumes O2 and generates CO2. Called “Dark Reac@ons” because the enzymes func@on in the absence of light IN VITRO. In Vivo, the Calvin Cycle only FUNCTIONS in the light in plants.

The Calvin Cycle (the Calvin Benson Bassham Cycle)

CO2 -> {CHO}

Biochemical Impera@ves for Plants

Glycolysis

Labelling 10 Sec Use of 14CO2 to label Intermediates

2-Dimensional This Layer Chromatography of labeled Intermediates

Labelling at 30 Sec

Labelling at 60 Sec

termediates forrned in the cycle depart from it on various patl'rrvnys to be con- verted to the er-rd products of photosyn- thesis. ['rom triose phosphate, for exam- ple, one seqllence of reactions leads to the six-carbon sugtrr glucose trnd the large family of carbohydrates.

Becanse the cycle had beer-r estab- lishecl prirnarily by experirnents with al- giie and the lenves of a few higher plants, it rv:rs irnpoltant to see whether or not the cycle prevailed thloughout the plant kingclom. Calvin and Louisir and Richnld Norris ctrrried out experiments with a wide variety of photosynthetic organ- isms. In every case, although they found variation in the arnounts of particular intennediates formed, the pattern was qualitatively the strme.

It also had to be shorvn that the path- way we had traced out is quantitatively the most important route of ctrrbon re- cluction in photosynthesis. To this er-rcl

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Martha Kirk and I ur-rdertook an inten- sive study of the kinetics of the florv of carbon in photosynthesis. Our stucly has helpecl to solve other genertrl problen-rs, particulrrrly the question of how carbon enters into the pathu,trys leading to the synthesis of proteirs nncl ftrts. The bio- logictrl n-rtrteri:rls fol this rvot'k irre sttp- plied by trn algae culture system uncler autornatic feedback cor.rtrol. L-r this ap- paratus \ve are irble to rnaintain the photosynthetic process in a steadv sttte, rvith rrutrients suppliecl at a constant rate ard u,ith temperature, clensity, salinity and acidity held rvithin nalrow liurits.

At the sttrrt of a nur rve inlect radio- active bicalbonate ion into the cultule medium along u,ith raclioirctive calbor.t dioxicle gas and so brir-rg the ratio of car- bon 14 to calbon 12 irnmediiitel.,, b its ffnal level in both the gas ancl the lirpricl phase. An turtornatic recordel lneilsul'es the rate trt u,hich calbon is absorbed by the photosynthesizing cells. We take sirmples every few seconcls and kill the cells irnn-rediately by irnmersir.tg them in alcohol. After u'e have chromartographed the photosynthetic intermeditrtes and rneasnlecl their radioactivity rve then plot the appearance of labelecl cai'bon

in each of these comporurds as * ftttrctiot.t of time.

By the end of three to five minutes, oul records shorv, irll the stable inter- rnediates of the cycle ale satut'atecl lvith calbon 14. Tnking the total umotttrt of carbon thus fixed ir-r cor.tpottnds artcl cornparing it "viih the rate of upttrke ofctrrbon ir-r the cultule, rve founcl that the cycle accounts for tnore thnn 70 per ce)lt of the tot:rl carbon fixed by the algae. A srnrrll but signiffcant amourrt is also t:rken up by the trddition of cnrbort dioxicle to a three-calbon compottnd, phosphoenolpyrttvic trcid, to give four- clrbon comportnds,

Frorn the earliest rvork u,ith carbon 14 in our laboratory, it had beert lpparerlt thtrt carbon dioxide fincls its way rather quickly into products othel thirlt carbo- hvdrates in the photosyr-rtl-resizilrg plant. This r':rs at variance 'uvith tltrclitiottal ideas trbout photosynthesis that regarded carbohyclrates as the sole olglnic prod- ucts of tl're process. It was itnportitnt trr ask, therefore, r,r''hethel f:rts and trrnitro acicls could be forn-red direcily fron the cycle irs products of its intern'rediates or' rvhether these noncarbohydrates rvere synthesized only frorn the carbohydrate end products of photosynthesis. Our kinetic stuclies shorv thnt certi"tirt amitttr acids rnust in<leed be forrned from thc interrnediates and must thelefore be le- garcled as true prodrtcts of photosynthe- sis. The irrniuo acid alanine, for exatnple, shou,s up labeled by carbon 14 at least as rapidly as any carbohydrate; it r.voultl be lnbeled rvith carbon 14 mrtch rnore slou,ly if it r'vere mirde from calbo- hvdr-ate, since the carbohydrirte u'oulcl hrrvc to be labeled first. lVe htrve bec'rr nble to show tlint more thrrn 30 per cent of the carbon takerr up bv the algae irr orrr steatl;-stitte systern is irtt'orpo- ratecl clirectly into irtnitio acicls. Thele is some evidence thtrt fats mirl' also lt iolrne<l as products of the cvcle.

The discovely tl-rat plants mtrke these other compounds as dilect proclucts oll pl-rotosynthesis lends new itrterest antl importance to the chloroplrrst, the sul> cellular corrp:rrtmelrt of greer.r cells that contair.rs pign-rents turd the rest of the photosynthetic tlpparattls. It has beerr knor'r,n for sonre tirre that this highly structured organelle is responsible for the nbsorption of light, the splitting of rvater and the forrnrrtion of the cofactors for carbon recluctiotr. N{ore recent stucl- ies have shorvn thtrt it is the site of the entire carbon-reduction cycle. Now the cl-rloroplast ernerges as a complete photo- synthetic factory for the productiou of just irbout everything necess:rry to the plarrt's glorvth iIn(l flrr(lior).

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CALVIN CYCLE (see illustraion on plges 90 md 91) rvas shorvn to be the most important route of carbon reduction in photosynthesis in studies by the author and Martha Kirk. As seen here, all stable intermediates of the cycle lrecome saturated rvith labeled carbon lvithin three to five minutes. Comparison rvith the rate of carbon uptake in the algae culture shorved that the cycle accounts for more than 70 per cent o{ the carbon fixed in compountls.

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Time Course of Labelling The First Intermediate Reaches its max labelling First.

Important! Know This!!!

Book keeping for Calvin Cycle For each 3 cycles, a net of one molecule of G3P is obtained

!!!!!!

Ru5P is Carboxylated & Then Split into Two Molecules of 3PG

Overview of the Dark Reactions

The Calvin Cycle

RuBP Carboxylase

“Rubisco”

RuBP carboxylase PDBid 1RLC

Dodecamer: 6 Large and 6 Small subunits As much at 50% of the soluble protein in leaves.

RuBP Carboxylase L Subunit

RuBP Carboxylase Mechanism

Free Energy Changes of the Calvin Cycle

Light-Activation Mechanism of FBPase and SBPase

Levels of Fructose diP and Sedoheptulose diP in light and dark

Regula@on of Calvin Cycle by pH, dithiols

Probable Mechanism of RuBP Carboxylase-Oxygenase

Rubisco Rxns

Photorespiration

Photorespira@on (not really respira@on)

Photorespiration

Photorespiration

The C4 Pathway

Transpira@on and Photosynthesis

The Phosphate Translocator Maintains P Homeostasis Transports C in/out of Chloroplast Transports NADH & ATP “Equivalents” in and out of Chloroplast 1:1 exchange of PO4 for Triose Phosphates

Professor Ludden thinks Phosphate Translocator is VERY COOL!

Consider the Potato!

The Phosphate Translocator Maintains P Homeostasis Transports C in/out of Chloroplast Transports NADH & ATP “Equivalents” in and out of Chloroplast 1:1 exchange of PO4 for Triose Phosphates

Professor Ludden thinks Phosphate Translocator is VERY COOL!

Starch Synthesis

In plants, ALL starch synthesis takes place in the PLASTID

Starch Synthesis

Sucrose Synthesis

Sucrose synthesis and breakdown occurs in the cytosol of plant cells.

Sucrose is the “transport sugar” of plants.