LON-CAPA MCB 229 Spring 2000: Metabolism: some anabolic pathways

		Quiz Me!	Metabolism: some anabolic pathways
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Last revised: Tuesday, February 29, 2000
Ch. 10 (p. 191-193; 196-203; 206-208) in Prescott et al, Microbiology, 4th Ed.
Note: These notes are provided as a guide to topics the instructor hopes to cover during lecture. Actual coverage will always differ somewhat from what is printed here. These notes are not a substitute for the actual lecture!
Copyright 2000. Thomas M. Terry

The Synthetic Needs of the Cell

Cell must make a wide variety of polymers and monomers

20 Amino Acids (AA) -----> proteins (-----> enzymes, permeases, cytochromes, etc.)
6-C sugar -----> amino sugars -----> peptidoglycan (-----> cell wall)
fatty acids + glycerol + phosphate-----> phospholipids (-----> membranes)
ribose & deoxyribose, purines & pyrimidines, phosphate -----> RNA & DNA

Conclusion: cell needs to synthesize lots of monomers -----> lots of polymers. Collectively called anabolism: synthesis of complex molecules from simple precursors. Must compete with catabolism for CHNOPS atoms, major use for energy generated by catabolism.

Amino Acid synthesis

Glutamic Acid Synthesis

alpha-ketoglutaric acid (KG) + NH₄⁺ + NADPH -----> glutamic acid + H₂O + NADP⁺
Notes:
(a) KG is the 5-C compound in the TCA cycle -- this reaction "siphons" KG out of TCA cycle to make an AA
(b) Enzyme is glutamic dehydrogenase; adds ammonium ion directly to substrate

Synthesis of other amino acids

Might imagine that all amino acids are made as glutamic acid is: add ammonium ion to keto group with NADPH.
But most amino acids actually synthesized by transamination reactions: swap an amino group from one amino acid (e.g., glutamic acid) with the keto group of another molecule.
Ex: glutamic acid (an AA) + oxalacetate (OAA) -----> alpha-ketoglutaric acid (KG) + aspartic acid (another AA)
Enzyme is a transaminase . Very common enzyme in AA metabolism

How to get NH₄+?

"Pirate" NH₂^- from pre-existing molecules This is the most cost-efficient strategy. Synthesis of amino groups requires energy
Assimilatory nitrate (NO₃^-) reduction. This is the most typical scenario. Nitrate is often available. Must be reduced by series of enzymes (dehydrogenases):

Ex: NO₃^- (nitrate) + NADPH ----------> NO₂^- (nitrite) + NADP⁺
NO₂^- (nitrite) -----[2H]----> -----[2H]----> -----[2H]----> NH₃ (ammonia) -------> NH₄⁺(ammonium ion)
Note: must distinguish assimilatory nitrate reduction from dissimilatory nitrate reduction; latter occurs during anaerobic respiration, nitrate is used as external electron acceptor, can form a variety of reduced products ranging from nitrite to dinitrogen gas. These are discarded as wastes, rather than assimilated into cell's organic molecules.

Nitrogen fixation
This is only carried out by certain bacteria. Some are free living (e.g. Azotobacter, some species of Clostridia, etc.); some are symbionts living in root nodules of certain plants (e.g. Rhizobium, Frankia, etc.)
Process is very expensive (even though overall G_o^' is negative) because of extremely high activation energy to split dinitrogen gas.
Reaction: N₂ ---[2H]---> HN=NH ---[2H]---> H₂N-NH₂ ---[2H]---> 2 NH₃
Note: each reduction requires ~ 4-5 ATP, so overall reduction requires ~12-15 ATP, as well as reducing power (different sources in different bacteria: NADPH, ferredoxin, others).
Enzyme required is nitrogenase (requires Molybdenum). Only works under anaerobic conditions, even though many nitrogen fixers are aerobic. Cell must create very reducing conditions in those regions where nitrogen fixation occurs. In some bacteria, have special "differentiated" cells that carry this out.

Amphibolic pathways

Note that amino acid (and other) synthesis makes use of molecules that are also involved in catabolism: KG, OAA, pyruvate, 3-phosphoglycerate, etc.
So it is not accurate to call glycolysis + TCA cycle strictly catabolic; a better term is amphibolic = both anabolic and catabolic functions can be served.

Anaplerotic Reactions

Consider cell growing with glucose as sole C-source: needs to break down glucose via glycolysis & TCA cycle to get Energy; but also needs to remove KG, OAA from TCA cycle to make AAs.
Imagine start with 100 glucose molecules ------> 200 pyruvates -----> 200 Acetyl-CoA ------> into TCA cycle.
But now remove 50 molecules from TCA cycle as KG to make amino acid glutamate; remove another 50 to make aspartate. Leaves only 100 OAA molecules to combine with next round of Acetyl-CoA.
Next step: another 100 glucose -----> 200 Acetyl-CoA ------> into TCA cycle. But now half of these Acetyl-CoA molecules can't enter TCA cycle, because only 100 OAA acceptor molecules waiting. Again, remove 50 KG, 50 OAA to make amino acids.
Next step: yet another 100 glucose -----> 200 Acetyl-CoA. But now, all TCA cycle molecules are gone (used to make AAs); zero OAA is left to combine with Acetyl-CoA.
Big problem!! What to do?
Solution: need a new type of reaction to replace missing TCA cycle intermediates. Called Anaplerotic (= replacement) reaction. What could it be?

Wood-Werkman reaction

First discovered by microbiologists/biochemists at Berkeley. Suggested that PEP (3-C molecule in glycolysis pathway just before pyruvate) could accept carbon dioxide (CO₂) using PEP carboxylase enzyme, form OAA (the 4-C molecule in TCA cycle that accepts Acetyl-CoA).
Enzyme has been isolated, has exactly these properties. Found in E. coli, other enteric bacteria.
Some organisms (e.g. yeasts) have different anaplerotic carboxylase enzyme, adds CO₂ to pyruvate (requires ATP) instead of to PEP (which has high energy phosphate, doesn't need additional ATP).
Net result: instead of breaking all glucose down to pyruvate and Acetyl-CoA, send some from PEP or pyruvate into TCA cycle by carboxylase reaction, bypassing Acetyl-CoA step. This reduces total yield of ATP, but allows TCA cycle to serve amphibolic needs.

How to make fatty acids?

Use Acetyl-CoA (2-C) as donor. See text for details.
NADPH provides reducing power to convert C-O groups into C-H₂ groups.
Fatty acids grow by successive addition of 2-C units.
Most biological fatty acids are between 12C and 24C in length, in 2C units. Most common are 16C and 18C long (in mesophiles).

How to make nucleotides for RNA & DNA?

Process is complicated; purines derive atoms from seven different molecules; pyrimidines originate from products of glutamine and aspartic acid.
Ribose and deoxyribose are made from pentose phosphate pathway (see notes on catabolism)
Phosphate is widely available in environment as inorganic ion; in addition, many organic molecules contain phosphate, can be made available by enzymes called phosphatases.

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