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Advance Biochemistry

Advance Biochemistry. Introduction. Goals To cover aspects of biochemistry unique and important to plants Sometimes will involve bacterial biochemistry To see some of the many biochemical pathways critical to plants To learn about regulation. Overall plan.

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Advance Biochemistry

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  1. Advance Biochemistry

  2. Introduction Goals • To cover aspects of biochemistry unique and important to plants • Sometimes will involve bacterial biochemistry • To see some of the many biochemical pathways critical to plants • To learn about regulation

  3. Overall plan • Cell and Cellular Constituents • Metabolism • Mode of regulation • Applied Biochemistry

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

  5. Cell • Unit from which living organisms are built • It consists of a plasma membrane surrounding the cytoplasm, in which a variety of structures may be present Two basics types of Living Cells Prokaryotic : Cells that have no internal, membrane-bounded structures and no clearly defined nucleus Eukaryotic : Cells that contain a nucleus surrounded by a membrane, the nuclear envelope

  6. Eukaryotic cells • They have developed an internal system of membranes that separates the cells into distinct areas, called organelle • The organelles have specific biochemical function and allow more ordered and directed metabolism to occur • Multicellular eukaryotic organisms have evolved cells with very specialized functions and structures which often associated in large numbers to form clearly identifiable tissue

  7. Component of eukaryotic cells • Plasma membrane • Cytoplasm • Nucleus • Cell walls • Ribosomes • Endoplasmic reticulum • Vacuoles and specialized vesicles • Mitochondria • Chloroplasts • Cytoskeleton

  8. Plasma membrane • a selectively permeable barrier that control the movement of molecules into and out of the cells • Transport of essential nutrient required for growth and metabolism • Hormone/receptor interactions • Cell recognition

  9. Cytoplasm • This is composed of the cytosol • Glycolysis • Gluconeogenesis • pentose phosphate pathways • polysaccharide breakdown • Complex lipid breakdown • Fatty acid synthesis • Protein breakdown • Amino acid synthesis

  10. Nucleus • DNA synthesis • RNA synthesis • RNA processing

  11. Cell walls • it determines to a great extent the morphology and to some extent the function of the cell • It may directly involved in regulating cell expansion

  12. Ribosomes • Responsible for the synthesis of proteins

  13. Endoplasmic reticulum • It is divided in two parts: rough and smooth • The 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 cell • In the smooth ER occurs fatty acid elongation and desaturation, complex of lipid synthesis and detoxification reactions • Of the layer of smooth ER stacked golgi apparatus which is the site of carbohydrate synthesis, glycoprotein synthesis, and packaging of cell product

  14. Vacuoles and specialized vesicles • vacuoles: • Stored materials separated from the main biochemical process of the cells • Pigment, toxic and waste material may be accumulate • Maintenance of turgor • Peroxisomes: • Fatty acids oxidation, • amino acids oxidation, • Photorespiration

  15. Mitochondria • TCA cycle • Fatty acid oxidation • Amino acid oxidation • gluconeogenesis • Synthesis of organelle protein

  16. Chloroplasts • Photosynthesis • Fatty acid synthesis • Complex lipid synthesis • Synthesis of some amino acids • Synthesis of organelle protein • Calvin cycle • Light reaction • Reduction of nitrate and sulphate • Part of photorespiration

  17. Methods of regulation • Properties of enzymes • Compartmentation • Gene expression

  18. Methods of regulation • Properties of enzymes • Affinity for substrate, inherent catalytic capacity • Feedback regulation/feedforward/loopgain • Allosteric effects, competitive versus non-competitive inhibition • Fructose 2,6-bisphosphate as an example • Redox control of enzymes (vicinal cysteines can become cystine) • pH and Mg regulation • Especially chloroplast enzymes

  19. Methods of regulation • Properties of enzymes (Post-translational regulation) • Phosphorylation • Protein kinases and phosphatases • Turns enzymes on or off, can affect sensitivity to effectors (SPS) • Fatty acids • Palmitic acid in a regulatory way, myristic acid is non-regulatory • Prenylation • Fanesylation (3 isoprenoids, 15 C) CaaX C-terminus • Geranylgeranylation (20 carbons) CaaL C-terminus • Fatty acids and prenylation anchors proteins to membranes or to other proteins

  20. Anchoring proteins to membranes Buchannan et al. (ASPB book) Fig. 1.10 page 9

  21. Methods of regulation • Cellular compartmentation • Hallmark of eukaryotic cells • Oxygen reactions mostly in mitochondria and chloroplasts • Chloroplasts – more generally plastids – are what make plants unique • Cell walls, vacuoles also distinctive but not unique • Plastids are biochemical powerhouses • I hope this course will leave you with an appreciation for the unique biochemistry of plants, and where in the cell it happens

  22. The family of plastids Buchannan et al. Fig. 1.44

  23. Endosymbiosis • Well accepted that chloroplasts and mitochondria were once free living bacteria • Their metabolism is bacterial (e.g. photosynthesis) • Retain some DNA (circular chromosome) • Protein synthesis sensitive to chloramphenicol • Cytosolic P synthesis sensitive to cycloheximide • Most genes transferred from symbiont to nucleus • Requires protein tageting

  24. Phylogenetic location of chloroplasts and mitochondria

  25. DNA for chloroplast proteins can be in the nucleus or chloroplast genome Buchannan et al. Fig. 4.4

  26. Import of proteins into chloroplasts Buchannan et al. Fig. 4.6

  27. Biochemistry inside plastids • Photosynthesis – reduction of C, N, and S • Amino acids, essential amino acid synthesis restricted to plastids • Phenylpropanoid amino acids and secondary compounds start in the plastids (shikimic acid pathway) • Site of action of several herbicides, including glyphosate • Branched-chain amino acids • Sulfur amino acids • Fatty acids – all fatty acids in plants made in plastids

  28. Biochemistry inside plastids • Carotenoids – source of vitamin A • Thiamin and pyridoxal, B vitamins • Ascorbic acid – vitamin C • Tocopherol – vitamin E • Phylloquinone (an electron accepttor in PS I – vitamin K)

  29. Photorespiration is highly compartmentalized Buchannan et al. Fig. 1.40

  30. Methods of regulation • Gene expression • Normally slow relative to metabolic control that will be discussed most of the time in this course • Allows metabolism to be changed in response to environmental factors • Transcriptional control most common • Sometimes variation in transcription rate not reflected in enzyme amount • Translational control also found • No change in mRNA levels but changes in protein amounts

  31. Gene structure relevant to metabolic regulation

  32. Promoters

  33. Exploring metabolism by genetic methods • Antisense – what happens when the amount of an enzyme is reduced • not clear how antisense works • Knockouts • Often more clear-cut since all of the enzyme is gone • Use of t-DNA, Salk lines • Overexpression • Use an unregulated version of the protein or express on a strong promoter • Sometimes leads to cosuppression • RNA interference • 21 to 26 mers seem very effective in regulating translation

  34. What do we expect for the reaction of metabolism to changes in amount of an enzyme? • Is subtracting 50% likely to give exactly the opposite result as adding 50%? • Are there threshholds? • Are there optimal amounts? • Are there compensatory pathways? • Are there compensatory regulatory mechanisms? • Kacser H, Porteous JW. Control of metabolism: what do we have to measure. Trends Biochem.Sci. 1987;12:5-14. • Koshland DE. Switches, thresholds and ultrasensitivity. Trends Biochem.Sci. 1987;12:225-9.

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