The 2ND EVENING EXAM will be held in 112 WALKER BUILDING at 6:30 PM on NOVEMBER 5, 1996. The format for this exam will be quite similar to the previous exam. You may need a calculator for this one, and you are welcome to bring one along if you feel the need.

1. Precursor metabolites and building blocks only made as they are needed--no excess secreted. HOW?

2. Label cells with ¹⁴C-glucose, and measure incorporation into amino acids. Repeat in presence of an amino acid, vitamin, purine, pyrimidine, etc. For most of these, no labeling will occur (There are a few amino acid exceptions to this rule: aspartate, serine, glutamate, proline, glycine, alanine, cysteine) This implies that cells can specifically shut off pathways. HOW?

3. Amino acid contents of proteins vary about 10-fold, (content of Ala or Gly about 10X that of Trp). All are synthesized by enzymes encoded by single genes. Does cell have different pool sizes for these? No, so HOW does cell regulate?

4. We have seen that cells must balance ATP/NAD(P)H production with precursor metabolite production. HOW?

5. Different organisms have preferred carbon/energy sources. Easily demonstrated by growing bacteria with two carbon/energy sources. One will be used preferentially. Examples: E. coli: glucose. Pseudomonas putida: succinate. HOW?

Requirement for adaptability and rapid, balanced growth probably forced the development of control mechanisms.

Since it is obvious that metabolic activities are regulated, the question becomes HOW.

There are basically 2 mechanisms to regulate metabolic activity of cells:

Regulation of Enzyme Activity (Chap. 11).

Regulation of Enzyme Synthesis (Chap. 12).

Both are controlled primarily by "ALLOSTERIC PROTEINS," proteins which bind small molecule LIGANDS (or EFFECTORS) or large molecule MODULATORS and can exist in more than 1 stable conformation. Allosteric proteins can be enzymes or proteins that affect enzyme synthesis (e.g., transcription activators or repressors).

1. Product Inhibition

2. Feedback Inhibition

3. Energy (& Redox) Charge Regulation

4. Covalent Modification

1. Product Inhibition. Products obviously chemically resemble substrates to some extent, and high concentrations of products can inhibit enzyme activity. Depending upon enzyme mechanism, binding of product to active site may be competitive or non-competitive with binding of substrate.

2. Feedback Inhibition/End-Production Inhibition

The word "INHIBITION" refers to inhibition of enzyme activity--has nothing to do with enzyme synthesis.

Typical, non-allosteric enzyme exhibits so-called hyperbolic kinetics when reaction velocity is plotted as a function of substrate concentration. Activity initially rises as [Substrate] increases, but eventually slows as active site becomes saturated with substrate. Adding additional substrate no longer increases the rate, and maximal velocity is achieved.

The substrate concentration that yields 0.5 v_max is a constant for each enzyme under defined conditions, and is the Michealis-Menten constant K_m.

Most key regulatory enzymes exhibit very different behavior: most exhibit sigmoidal kinetics, with POSITIVE COOPERATIVITY, and most are multi-subunit enzymes. Allosteric enzymes have binding sites for effectors that are not at the active site, but (usually; in a few cases it is v_max that is altered) affect the affinity of the enzyme for one of its substrates.

Phosphofructokinase is a key control step in glycolysis. This tetrameric enzyme is stimulated by the allosteric affectors AMP and ADP, but is inhibited by ATP. When AMP+ADP conc. is high, ATP concentrations must be low, and the enzyme has a much lower K_m for fructose-6-P (see below). Phosphoenolpyruvate also acts as an allosteric inhibitor of phosphofructokinase. Effector binding alters the conformation of the tetramers significantly. When AMP/ADP binds to allosteric sites, the enzyme subunits occupy less space and rotate relative to one another.

Textbook (p. 307) also discusses Aspartate Transcarbamylase. ATCase is a very large and complex enzyme: 6 catalytic large subunits, arranged in two groups of 3 (ring-shaped trimers). There are also 6 regulatory small subunits, organized into regulatory dimer pairs and sandwiched between the two catalytic trimer rings, that have the binding sites for the allosteric effectors CTP and ATP. This is a key control enzyme for biosynthesis of pyrimidines (U, T, C) and catalyzes the reaction of carbamoyl phosphate + aspartate to yield carbamoyl aspartate. Figure 1, page 307 of text shows that CTP acts a feedback inhibitor of the enzyme, shifting apparent K_m to higher values. The purine ATP also acts as a positive effector of this same enzyme, shifting the K_m to much lower values and changing the kinetics to be much more hyperbolic. This effect allows the synthesis of purines (A+G) to be balanced with synthesis of pyrimidines (U, T, C).

1. usually occur at metabolic branch points (catalyze the first commited step in a pathway).

2. are usually multi-subunit enzymes with complex kinetic behavior

3. usually catalyze reactions that are physiologically irreversible (far from equilibrium).

1. Simple feedback inhibition.

2. Sequential feedback inhibition.

3. Concerted or Cumulative Feedback

4. Isofunctional enzymes.

5. Inhibition + activation systems.

In simple feedback inhibition, the end product inhibits the enzyme catalyzing the first commited step in the pathway leading to that product.

Sequential feedback is observed in some branched pathways. Inhibition of the enzymes at the branchpoints causes intermediate to accumulate, and this intermediate then feeds back to inhibit the enzyme catalyzing the first commited step for the entire pathway.

Concerted or cumulative feedback is an alternative solution for the same problem. The enzyme catalyzing the first commited step is controlled by both end products; it does not have to be equally sensitive to each.

Isofunctional enzymes for the first commited step is another possible solution. By having multiple enzymes for this step, each can be sensitive to a single endproduct.

Finally, many pathways are subject to both positive and negative controls, as in the case of aspartate transcarbamylase. This allows coordinate control and more global regulation to occur.

Figure 4 (p. 311of text) shows the major control points in centeral pathways of the fueling reactions (glycolysis, pentose-phosphate pathway, and TCA cycle). Primary regulators are AMP, ADP, phosphoenolpyruvate, Fructose 1,6-bisphosphate, Acetyl-CoA, NADH, aspartate, and a-ketoglutarate.

ATP, ADP and AMP are all involved in control of ATP synthesis, since biosynthesis and assembly involves cyclic interconversions of all three adenylate compounds (see below).

Some enzymes respond to absolute concentration, but most respond to ratios. Dan Atkinson introduced the concept of ENERGY CHARGE in 1968 to summarize the energy status of a cell. It is a measure of the relative concentration of high-energy phospho-anhydride bonds available in the adenylate pool.

Although mathematically the energy charge can vary between 0 (all AMP) and 1 (all ATP), bacteria typically maintain this pool in the range 0.87 to 0.95, and this does not vary as a function of growth rate. Abruptly shifting E. coli from aerobic conditions to anaerobic conditions has very little affect on the energy charge value. At an energy charge of 0.5, a cell is operationally dead. NOTE: enzymes cannot respond to energy charge, but produce an integrated response based upon the concentrations of total adenylates. Energy charge is a useful mathematical tool to predict and describe the observed responses of many adenylate-regulated enzymes.

Phosphofructokinase, citrate synthase, pyruvate dehydrogenase, and isocitrate dehydrogenase are all enzymes that respond like ATP-regenerating enzymes (includes some enzymes of glycolysis, pentose-phosphate pathway, and TCA cycle), while aspartate kinase, phosphoribosyl-ATP pyrophosphorylase, and phosophoribosyl-pyrophosphate synthetase behave as ATP utilizing enzymes (many anabolic enzymes).

Overproduction of ATP may in some instances occur, and this can be dissipated by so-called FUTILE CYCLES, cycles for which the net reaction is the hydrolysis of ATP to ADP or AMP. Clearly such reactions must be carefully regulated, or ATP would be wasted.

Similar concepts can be introduced for NADH and NADPH. The Catabolic Reduction Charge and the Anabolic Reduction Charge, respectively.

Recall: NADH is primarly used for energy production (catabolic reactions) and NADPH is primarily used in biosynthetic (anabolic) reactions. The CRC value is maintained low, since the oxidized form is the primary reactant. For anabolic reactions, the reduced form NADPH is usually used, so ARC value is about 10-fold higher. Regulation of these values is not well understood. TRANSHYDROGENASE can catalyze exchange between the two pools; reduction of NADP⁺ by NADH requires a protonmotive force (1 H⁺ consumed for 2 electrons transferred). However, transhydrogenase mutants still grow well, so other parameters must be involved in controlling these ratios.

Covalent modification as a control mechanism is very common in eucaryotes, in which phosphorylation is frequently used to modulate activities of enzymes. Although probably less common in bacteria, there are nonetheless many examples of covalent modification that occur.

Table 1, p. 315 lists several enzymes/proteins and the post-translational modifications that occur. A mechanism not listed, but one that is certainly important is TURNOVER TIME. Proteolytic degradation of a protein is a very important mechanism for altering the enzyme activity/metabolic potential of cells. It is a "covalent" modification, since the peptide bonds linking amino acids are hydrolyzed.

You have already heard and seen how phosphorylation and methylation function in two-component regulatory systems and the related chemotaxis signaling. Additional examples are acetylation of ribosomal proteins and methylation of other proteins to modulate activities. Additional examples will be discussed briefly: addition of nucleotide units (adenylylation, uridylylation, and ADP-ribosylation) and phosphorylation of the enzyme isocitrate dehydrogenase.

Dinitrogen gas (N₂) reduction is energetically very expensive, requiring much ATP and reducing power. Nitrogenase activity in a number of bacteria (especially purple bacteria) is regulated by ADP-ribosylation of the dinitrogenase reductase at a specific arginine residue (the modifcation has the structure Arg-ribose-P-P-ribose-Adenine). Ammonia and darkness stimultate this reaction; the ADP-ribose donor is NAD⁺; the modifying group is removed by a separate enzyme that is requires ATP and metal ions. Low NH₄⁺ stimulates the removal enzyme and re-activates nitrogenase for reduction of N₂ gas to NH₃. A similar modification of some glutamine synthetases has been reported but details remain limited.

The glyoxylate bypass consists of two reactions that allow the net conversion of Acetyl-CoA into metabolic intermediates. The glyoxylate cycle is required for growth of bacteria on acetate or fatty acids. Isocitrate lyase and malate synthase are required, encoded by the aceA and aceB genes, respectively (induced by acetate). Isocitrate lyase cleaves isocitrate yielding succinate and glyoxylate; malate synthase produces malate from condensation of glyoxylate with Acetyl-CoA. These steps bypass the oxidative steps of the TCA cycle that would otherwise convert acetate into CO₂. Regulation of the flow of carbon through the TCA cycle and the glyoxylate shunt is modulated by phosphorylation of isocitrate dehydrogenase (IDH) by AceK protein. When cells are grown in glucose, a low amount of isocitrate is produced; IDH with high v_max (320 mM/min) and very low K_m (8 mM) converts all isocitrate into a-ketoglutarate CO₂ and ultimately converts acetyl-units primarily to CO₂. During growth on acetate, phosphorylation "inactivates" IDH, causing a 4-fold reduction in v_max; increased production of isocitrate (about 5.5-fold more than in glucose grown-cells) and the decrease in v_max combine to produce an increase in isocitrate levels in cells. Flux through isocitrate lyase, which has a relatively high K_m for isocitrate (0.6 mM), increases. Flux through the "inactive" IDH actually increases in cells to match the v_max of IDH-P_i (about 80 mM/min). This result, which is counterintuitive, is required since cells must produce much more isocitrate when growing in acetate in order to have precursors for hexose and pentose (building block) biosynthesis as well as acetyl units for oxidation and energy production. Activity in a test tube and "activity" (flux) in a cell are two different things. In a cell activity also depends on availability of the substrate, and that may change dramatically as a function of nutrient availability in the environment.

Nitrogen makes up ~14% of the cell mass. As you saw previously, glutamine synthetase plays an important role in NH₃ assimilation. At low NH₃ concentrations, it is the primary uptake route, but consumes ATP, so cells regulate extensively and rapidly. See Figure 23.

Glutamine synthetase from E. coli has 12 identical subunits, arranged into two ring-shaped hexamers. Each subunit has a mass of about 50 kDa. Enzyme is feedback inhibited by 9 small molecules, most of which obtain nitrogen from glutamine: carbamoyl-phosphate, glucosamine-6-phosphate, tryptophan, histidine, alanine, serine, glycine, CTP and AMP. Sensitivity to feedback inhibitors varies with growth conditions. The enzyme from nitrogen-limited cells is less sensitive to feedback than the enzyme isolated from nitrogen-replete cells. This is due to covalent modification of the enzyme. Enzyme activity/sensivity is modulated by addition of AMP (adenyl group) to a specific tyrosine on each subunit. Degree of modification can range from 1 to 12 subunits modified; enzyme becomes progressively less active and more sensitive to feedback as it becomes more modified.

Figure 24 shows the regulatory scheme for GS.

GS-adenylylated and GS-deadenylylated are really 2 different enzymes. In nitrogen-rich media, GS is adenylylated, present in lower concentration (through transcriptional controls), and is very sensitive to feedback inhibition. It is primarily used for glutamine synthesis, not for NH₃ assimilation. In nitrogen-limited conditions, GS is present in higher amounts in cells, is deadenylylated, and less sensitive to feedback inhibition. Under these conditions, it is primarily used for NH₃ assimilation.

Enzyme activity control circuitry requires 3 proteins:

1. Adenyl transferase (ATase = GlnE)

2. Uridyl transferase (UTase/UR = GlnD)

3. P_II protein = GlnB

The P_II protein is a trimer, which can be uridylylated on each of its three subunits by UTase. Under low glutamine/a-ketoglutarate ratio conditions, UTase uridylylates P_II, which stimulates the removal of AMP groups by ATase, activating GS. At high glutamine/a-ketoglutarate ratio conditions, UTase removes uridyl groups from P_II, which interacts with ATase to stimulate adenylylation of GS, rendering the enzyme less active. The dual controls allows signal amplification and more sensitivity. Addtionally, the P_II protein can (and does) regulate other cellular processes.

This scheme is not universal. For example, in some bacteria the P_II protein is regulated by phosphorylation rather than uridylylation, and GS is not adenylylated but ADP-ribosylated. Molecular-level details of other systems are not as advanced as for enteric bacteria.