Advance Biochemistry

Advance Advance BiochemistryBiochemistry

IntroductionIntroduction

GoalsGoals• To cover aspects of biochemistry unique and important to plantsTo cover aspects of biochemistry unique and important to plants

Sometimes will involve bacterial biochemistrySometimes will involve bacterial biochemistry• To see some of the many biochemical pathways critical to plantsTo see some of the many biochemical pathways critical to plants• To learn about regulationTo learn about regulation

Overall planOverall plan

Cell and Cellular ConstituentsCell and Cellular Constituents MetabolismMetabolism

Mode of regulation Mode of regulation Applied BiochemistryApplied Biochemistry

http://www.sigmaaldrich.com/img/assets/4202/sigma_metabolic_path-new.pdf

Cell Cell Unit from which living organisms are builtUnit from which living organisms are built

It consists of a plasma membrane surrounding the cytoplasm, in It consists of a plasma membrane surrounding the cytoplasm, in which a variety of structures may be presentwhich a variety of structures may be present

Two basics types of Living CellsTwo basics types of Living Cells

Prokaryotic : Cells that have no internal, membrane-bounded Prokaryotic : Cells that have no internal, membrane-bounded structures and no clearly defined nucleusstructures and no clearly defined nucleus

Eukaryotic : Cells that contain a nucleus surrounded by a Eukaryotic : Cells that contain a nucleus surrounded by a membrane, the nuclear envelope membrane, the nuclear envelope

Eukaryotic cells Eukaryotic cells They have developed an internal system of membranes that They have developed an internal system of membranes that

separates the cells into distinct areas, called organelleseparates the cells into distinct areas, called organelle The organelles have specific biochemical function and allow more The organelles have specific biochemical function and allow more

ordered and directed metabolism to occurordered and directed metabolism to occur Multicellular eukaryotic organisms have evolved cells with very Multicellular eukaryotic organisms have evolved cells with very

specialized functions and structures which often associated in large specialized functions and structures which often associated in large numbers to form clearly identifiable tissuenumbers to form clearly identifiable tissue

Component of eukaryotic cellsComponent of eukaryotic cells Plasma membranePlasma membrane

CytoplasmCytoplasm NucleusNucleus

Cell wallsCell walls RibosomesRibosomes

Endoplasmic reticulumEndoplasmic reticulum Vacuoles and specialized vesiclesVacuoles and specialized vesicles

MitochondriaMitochondria ChloroplastsChloroplasts CytoskeletonCytoskeleton

Plasma membranePlasma membrane a selectively permeable barrier that control the movement of a selectively permeable barrier that control the movement of

molecules into and out of the cellsmolecules into and out of the cells Transport of essential nutrient required for growth and metabolismTransport of essential nutrient required for growth and metabolism

Hormone/receptor interactionsHormone/receptor interactions Cell recognition Cell recognition

CytoplasmCytoplasm

This is composed of the cytosolThis is composed of the cytosol GlycolysisGlycolysis

GluconeogenesisGluconeogenesis pentose phosphate pathwayspentose phosphate pathways

polysaccharide breakdownpolysaccharide breakdown Complex lipid breakdownComplex lipid breakdown

Fatty acid synthesisFatty acid synthesis Protein breakdownProtein breakdown

Amino acid synthesisAmino acid synthesis

NucleusNucleus

DNA synthesisDNA synthesis RNA synthesisRNA synthesis

RNA processingRNA processing

Cell wallsCell walls

it determines to a great extent the morphology and to some extent the it determines to a great extent the morphology and to some extent the function of the cellfunction of the cell

It may directly involved in regulating cell expansionIt may directly involved in regulating cell expansion

RibosomesRibosomes

Responsible for the synthesis of proteinsResponsible for the synthesis of proteins

Endoplasmic reticulumEndoplasmic reticulum It is divided in two parts: rough and smooth It is divided in two parts: rough and smooth

The rough ER is due to the present of ribosomes required for the synthesis of proteinThe rough ER is due to the present of ribosomes required for the synthesis of protein it is the site of synthesis of proteins destined to be secreted from the cellit is the site of synthesis of proteins destined to be secreted from the cell

In the smooth ER occurs fatty acid elongation and desaturation, complex of lipid In the smooth ER occurs fatty acid elongation and desaturation, complex of lipid synthesis and detoxification reactionssynthesis and detoxification reactions

Of the layer of smooth ER stacked golgi apparatus which is the site of carbohydrate Of the layer of smooth ER stacked golgi apparatus which is the site of carbohydrate synthesis, glycoprotein synthesis, and packaging of cell product synthesis, glycoprotein synthesis, and packaging of cell product

Vacuoles and specialized vesiclesVacuoles and specialized vesicles

vacuoles:vacuoles: Stored materials separated from the main biochemical process of the cellsStored materials separated from the main biochemical process of the cells

Pigment, toxic and waste material may be accumulatePigment, toxic and waste material may be accumulate Maintenance of turgorMaintenance of turgor

Peroxisomes:Peroxisomes: Fatty acids oxidation, Fatty acids oxidation, amino acids oxidation, amino acids oxidation,

PhotorespirationPhotorespiration

MitochondriaMitochondria TCA cycleTCA cycle

Fatty acid oxidationFatty acid oxidation Amino acid oxidationAmino acid oxidation

gluconeogenesisgluconeogenesis Synthesis of organelle proteinSynthesis of organelle protein

ChloroplastsChloroplasts PhotosynthesisPhotosynthesis

Fatty acid synthesisFatty acid synthesis Complex lipid synthesisComplex lipid synthesis

Synthesis of some amino acidsSynthesis of some amino acids Synthesis of organelle proteinSynthesis of organelle protein

Calvin cycleCalvin cycle Light reactionLight reaction

Reduction of nitrate and sulphateReduction of nitrate and sulphate Part of photorespirationPart of photorespiration

Methods of regulationMethods of regulation

Properties of enzymesProperties of enzymes CompartmentationCompartmentation Gene expressionGene expression


Properties of enzymesProperties of enzymes• Affinity for substrate, inherent catalytic capacityAffinity for substrate, inherent catalytic capacity• Feedback regulation/feedforward/loopgainFeedback regulation/feedforward/loopgain• Allosteric effects, competitive versus non-competitive Allosteric effects, competitive versus non-competitive

inhibitioninhibition Fructose 2,6-bisphosphate as an exampleFructose 2,6-bisphosphate as an example

• Redox control of enzymes (vicinal cysteines can Redox control of enzymes (vicinal cysteines can become cystine)become cystine)

• pH and Mg regulationpH and Mg regulation Especially chloroplast enzymesEspecially chloroplast enzymes

Methods of regulationMethods of regulation Properties of enzymes (Post-translational regulation)Properties of enzymes (Post-translational regulation)

• Phosphorylation Phosphorylation Protein kinases and phosphatasesProtein kinases and phosphatases Turns enzymes on or off, can affect sensitivity to effectors (SPS)Turns enzymes on or off, can affect sensitivity to effectors (SPS)

• Fatty acidsFatty acids Palmitic acid in a regulatory way, myristic acid is non-regulatoryPalmitic acid in a regulatory way, myristic acid is non-regulatory

• PrenylationPrenylation Fanesylation (3 isoprenoids, 15 C) CaaX C-terminusFanesylation (3 isoprenoids, 15 C) CaaX C-terminus Geranylgeranylation (20 carbons) CaaL C-terminusGeranylgeranylation (20 carbons) CaaL C-terminus

• Fatty acids and prenylation anchors proteins to membranes Fatty acids and prenylation anchors proteins to membranes or to other proteinsor to other proteins

Anchoring proteins to Anchoring proteins to membranesmembranes

Buchannan et al. (ASPB book) Fig. 1.10 page 9


Cellular compartmentationCellular compartmentation• Hallmark of eukaryotic cellsHallmark of eukaryotic cells• Oxygen reactions mostly in mitochondria and Oxygen reactions mostly in mitochondria and

chloroplastschloroplasts• Chloroplasts – more generally plastids – are Chloroplasts – more generally plastids – are

what make plants uniquewhat make plants unique Cell walls, vacuoles also distinctive but not uniqueCell walls, vacuoles also distinctive but not unique Plastids are biochemical powerhousesPlastids are biochemical powerhouses

I hope this course will leave you with an I hope this course will leave you with an appreciation for the unique biochemistry appreciation for the unique biochemistry of plants, and where in the cell it happensof plants, and where in the cell it happens

The family of plastidsThe family of plastids

Buchannan et al. Fig. 1.44

EndosymbiosisEndosymbiosis

Well accepted that chloroplasts and Well accepted that chloroplasts and mitochondria were once free living bacteriamitochondria were once free living bacteria

Their metabolism is bacterial (e.g. Their metabolism is bacterial (e.g. photosynthesis)photosynthesis)

Retain some DNA (circular chromosome)Retain some DNA (circular chromosome)• Protein synthesis sensitive to chloramphenicolProtein synthesis sensitive to chloramphenicol• Cytosolic P synthesis sensitive to cycloheximideCytosolic P synthesis sensitive to cycloheximide

Most genes transferred from symbiont to Most genes transferred from symbiont to nucleusnucleus• Requires protein tagetingRequires protein tageting

PhylogenetiPhylogenetic location of c location of chloroplasts chloroplasts

and and mitochondrimitochondri

aa

DNA for chloroplast proteins DNA for chloroplast proteins can be in the nucleus or can be in the nucleus or

chloroplast genomechloroplast genome


Import of Import of proteins proteins

into into chloroplaschloroplas

tsts


Biochemistry inside plastidsBiochemistry inside plastids

Photosynthesis – reduction of C, N, and SPhotosynthesis – reduction of C, N, and S Amino acids, essential amino acid synthesis Amino acids, essential amino acid synthesis

restricted to plastidsrestricted to plastids• Phenylpropanoid amino acids and secondary Phenylpropanoid amino acids and secondary

compounds start in the plastids (shikimic acid compounds start in the plastids (shikimic acid pathway)pathway)

• Site of action of several herbicides, including Site of action of several herbicides, including glyphosateglyphosate

• Branched-chain amino acidsBranched-chain amino acids• Sulfur amino acidsSulfur amino acids

Fatty acids – all fatty acids in plants made in Fatty acids – all fatty acids in plants made in plastidsplastids

Biochemistry inside plastidsBiochemistry inside plastids

Carotenoids – source of vitamin ACarotenoids – source of vitamin A Thiamin and pyridoxal, B vitamins Thiamin and pyridoxal, B vitamins Ascorbic acid – vitamin CAscorbic acid – vitamin C Tocopherol – vitamin E Tocopherol – vitamin E Phylloquinone (an electron accepttor Phylloquinone (an electron accepttor

in PS I – vitamin K)in PS I – vitamin K)

Photorespiration is highly Photorespiration is highly compartmentalizedcompartmentalized



Gene expressionGene expression• Normally slow relative to metabolic control that Normally slow relative to metabolic control that

will be discussed most of the time in this coursewill be discussed most of the time in this course• Allows metabolism to be changed in response Allows metabolism to be changed in response

to environmental factorsto environmental factors• Transcriptional control most commonTranscriptional control most common

Sometimes variation in transcription rate not Sometimes variation in transcription rate not reflected in enzyme amountreflected in enzyme amount

• Translational control also foundTranslational control also found No change in mRNA levels but changes in protein No change in mRNA levels but changes in protein

amountsamounts

Gene structure relevant to metabolic Gene structure relevant to metabolic regulationregulation

PromotersPromoters

Exploring metabolism by Exploring metabolism by genetic methodsgenetic methods

Antisense – what happens when the amount of Antisense – what happens when the amount of an enzyme is reducedan enzyme is reduced• not clear how antisense worksnot clear how antisense works

KnockoutsKnockouts• Often more clear-cut since all of the enzyme is goneOften more clear-cut since all of the enzyme is gone• Use of t-DNA, Salk linesUse of t-DNA, Salk lines

OverexpressionOverexpression• Use an unregulated version of the protein or Use an unregulated version of the protein or

express on a strong promoterexpress on a strong promoter• Sometimes leads to cosuppressionSometimes leads to cosuppression

RNA interferenceRNA interference• 21 to 26 mers seem very effective in regulating 21 to 26 mers seem very effective in regulating

translationtranslation

What do we expect for the What do we expect for the reaction of metabolism to reaction of metabolism to changes in amount of an changes in amount of an

enzyme?enzyme? Is subtracting 50% likely to give exactly the Is subtracting 50% likely to give exactly the

opposite result as adding 50%?opposite result as adding 50%? Are there threshholds?Are there threshholds? Are there optimal amounts?Are there optimal amounts? Are there compensatory pathways?Are there compensatory pathways? Are there compensatory regulatory mechanisms?Are there compensatory regulatory mechanisms? Kacser H, Porteous JW. Control of metabolism: Kacser H, Porteous JW. Control of metabolism:

what do we have to measure. Trends Biochem.Sci. what do we have to measure. Trends Biochem.Sci. 1987;12:5-14.1987;12:5-14.

Koshland DE. Switches, thresholds and Koshland DE. Switches, thresholds and ultrasensitivity. Trends Biochem.Sci. 1987;12:225-ultrasensitivity. Trends Biochem.Sci. 1987;12:225-9.9.

Advance Biochemistry

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