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Learning objectives associated with Winter_2021_Bis2A_Facciotti_Reading_16
• Describe the mechanism of allosteric regulation and provide examples of how this mechanism can be used to tune the flow of molecules in a metabolic pathway.
• In a given metabolic pathway, think about: why you might need regulation(ex: conservation of resources), what information will you use to regulate (ex: [metabolite]), where does it make sense to regulate (ex: branch points, essential steps with large -deltaG, rate-limiting steps), by what mechanism might you use to regulate this pathway (how), and what can/should do the regulating.
An introduction to metabolic regulation
We can describe cellular metabolic pathways as sets of interconnected enzyme-catalyzed chemical reactions. Together these pathways form complex networks that collectively guide the flow of molecules and energy throughout a cell by breaking down, building, and rearranging the atoms and molecules of nature into their many different forms.
The collective activity of metabolic pathways allows organisms to eat food (e.g. atoms and molecules) from their environments and to convert that food at a molecular level into the specific forms of matter the organism needs to support life. Metabolic pathways also allow organisms to harvest energy from the environment and to move that energy to places in their cells that need energy to get work done.
Understanding how metabolic pathways help biological systems reorganize matter and energy is therefore fundamental to our understanding of biology from the smallest cellular scale to the functioning of connected global ecosystems and their interactions with the non-living environment.
Metabolic decision making, sensors and switches
Many living systems have complex metabolic pathways that enable them to make and process many different types of molecules. While these complex pathways likely provide an organism with selective advantage not all pathways need to be functioning at the same levels all of the time.
If, for instance, a unicellular organism is able to find and eat plenty of the amino acid tryptophan from its environment, the organism’s metabolic pathways that synthesize tryptophan do not need to be highly active. Running metabolic pathways that don’t need to be active wastes both the atoms that pass through the pathways and the energy used in the creation of molecules that aren’t needed at a particular time. Moreover, since most living systems do not exist in resource-rich environments, atoms and energy must be carefully distributed to cellular functions that need them most, only when they are needed. Resources can then be converted into other forms when they are needed elsewhere in the cell.
To be competitive, the cell must be frugal with resources and thus control how and when different metabolic pathways are active. Controlling the flow of materials through metabolic pathways requires mechanisms for decision making - we use the anthropomorphic word “decision” to imply that the cell seemingly knows how to make choices, not to suggest that it has a brain. For example, the cell in the example above needs a way to know that tryptophan is in abundance and then to use that information to switch off the biosynthetic pathway for tryptophan biosynthesis. Concomitantly, the cell can choose to use the metabolic precursors for tryptophan biosynthesis for another purpose, activating enzymes in other pathways that can use those same precursor molecules. This kind of sensing and control is required to simultaneously coordinate the flow of biomolecules and energy throughout all of cellular metabolism. This means that there must be molecular sensors and switches for controlling metabolism distributed throughout the cell.
Figure 1. Overview of the major connections between core metabolic pathways in eukaryotes.
Attribution:Chakazul / CC BY-SA (https://creativecommons.org/licenses/by-sa/4.0) <https://upload.wikimedia.org/wikipedia/commons/6/6e/Metabolic_Metro_Map.svg>
Our daily lives are full of similar experiences and concepts that can inform how we think about metabolic control in biology. Our homes have all sorts of sensors and switches for managing energy. We have light switches in every room to control the flow of electricity and conversion of energy through light bulbs. Our stoves have switches on them to control energy use on each burner. Thermostats in the home sense temperature and use that information to adjust the activity of our air conditioners and/or heaters. Some of these switches are bistable, they turn things fully “on” or “off” (e.g. most light switches). Other switches are continuous (e.g. dial switches on a stove, the volume knob on the radio). They allow the user to control energy use along a gradient.
The hierarchical nature of regulatory systems
Another useful concept to draw from looking at how we manage energy in our homes is to note the hierarchical nature of the control systems we have engineered for our living spaces. Nearly all homes/apartments have circuit breakers that can help control the flow of power from an energy provider into the home. In a circuit breaker box one usually finds a master switch that can turn power on or off for the entire home. One can usually also find switches that control power to individual rooms or parts of the house (e.g. the kitchen, the bathrooms). Within each room, light switches mounted on the wall typically allow the resident to control energy flow to all or just some of the outlets in the wall. Finally, each device plugged into the wall has its own “on”/“off” switch. Many devices, like a radio, have additional switches on them that allow individualized energy use management. As we develop our understanding of regulation in biology, we will see that Nature has also evolved conceptually similar hierarchical control structures (though typically more interconnected) to help manage metabolism and various other cellular functions. Some signaling molecules target and regulate very specific control points - a single enzyme. Other molecular signals target many control points simultaneously (many enzymes can be coordinately regulated). The interplay between specific control signals and broader control signals creates the ability to respond to big changes quickly and also to fine tune responses in the cell in a very detailed way.
Figure 2. A schematic of a household wiring scheme showing hierarchical control of power to rooms, lights and devices. The master switch can turn off all power. Sub-switches can control power to individual rooms. Meanwhile, room-level switches can control power to parts of the room. Finally, switches on devices can control power at an individual device level. This serves as an analogy for some features of metabolic and cellular regulation.
Attribution: Original work - Marc T. Facciotti
The Who, What, When, Where, Why and How of Regulation
We structure the rest of this discussion of metabolic regulation by using a formalism often associated with journalism. We examine answers to the questions who, what, when, where, why and how <https://en.wikipedia.org/wiki/Five_Ws>.
To some degree, we have made the argument about why regulation is necessary above. Largely, regulation in metabolism is involved in making decisions about how to best manage limited resources - the atoms that make up biomolecules, the biomolecules themselves, and the energy needed to build new molecules and/or carry out cellular functions. Good decision-making leads to more efficient resource utilization and by consequence we suspect higher evolutionary fitness.
Who to regulate?
When we consider the regulation of metabolism and metabolic pathways, the key control points are the enzymes that catalyze individual reactions. In the simplest terms, increasing the abundance of a specific enzyme and/or increasing its activity (how fast it catalyzes a reaction) can increase the flow of molecules through that step of a pathway. By contrast, decreasing the abundance of an enzyme and/or lowering its activity will reduce the amount of material flowing through that step in a pathway.
Where should regulation be exercised?
If enzymes and their relative abundance are good targets of regulation we can now ask whether some steps in metabolic pathways are more important to regulate than others. The answer to this question is yes and can be largely summarized by finding enzymes that belong to one or more of three classes: (a) enzymes that catalyze steps at branch points in a pathway; (b) enzymes that catalyze rate-limiting reactions; and (c) enzymes that catalyze so-called irreversible reactions (i.e. reaction with a large negative ∆G). We’ll examine the logic behind each.
Enzymes that catalyze reactions at branch points: Branch points in metabolic reactions are points in a metabolic pathway in which a compound can be used as a substrate in two or more different biochemical reactions. The cell must “decide” which of the two or more pathways to direct the biomolecule to. This is no different than the everyday experience we each time we encounter an intersection in the road we’re traveling on - we must decide whether to continue straight or turn down a different path. We can’t do both at the same time. Another everyday example might be in the construction of a water distribution pipeline in a garden with two or more planting beds. Depending on what is planted in each bed the gardener may wish to tune the flow of water to each bed differently and would do so by opening and closing valves at the branch points of the watering system.
Enzymes that catalyze rate-limiting steps in a pathway: A rate-limiting step in a metabolic pathway is defined as the reaction that determines how fast the overall pathway overall can convert input into output. This is almost always the slowest step in the pathway. Regulating this step by either slowing or speeding flow of metabolites through it can change the flow for the entire pathway. By analogy, you can think of a 5-lane highway that needs to constrict to 3 lanes over a bridge. While the highway may widen back up to 5 lanes after the bridge, the maximum rate of traffic flow before the bridge and after the bridge is limited by how many cars can get across the bridge at any one time. Narrowing or widening the bridge will have a major impact on traffic flow both before and after.
Enzymes that catalyze irreversible reactions: Reactions that have a large negative ∆G are often called irreversible reactions. These reactions are also often considered to be “commitment” steps in a biochemical pathway because it is difficult to reverse the reaction once it is done. Typically, different enzymes and external energy sources are required to run the reaction in the endergonic direction. We can also draw upon an analogy here to understand why these are important decision points by thinking about decisions in life that are hard to take back once they are made. Consider, for instance, the act of buying a used car or other equivalent item of value that you might need, or care to have. Few people would consider buying such an item sight unseen and without asking questions, doing some research, and maybe even getting a professional opinion on the condition of the car or other item. If the car/item is still functional and well-maintained the purchase could work out fantastically well. On the other hand, if the car/item has some defects or in need of repair, the purchase may not end well at all. The decision to buy the car/item must be carefully considered BEFORE the purchase. After you hand over the money there is typically no opportunity to changing your mind – the sale if final - and you will need to live with the consequences of that decision, good or bad. The same idea applies to reactions with large negative ∆G. Once the decision has been made to catalyze the reaction, that decision cannot be easily reversed. Therefore, the cell must evolve mechanisms that help make these decisions carefully on the basis of good information.
In many metabolic pathways, particularly those that are as interconnected to other pathways as glycolysis, the picture is - of course - more complicated as each these three types of control points are each used for regulation and it is too simplistic to simply state that there is a single point in the pathway that controls flow. Rather, it is the combination and interplay of regulatory sites and regulatory mechanisms that ultimately determines flow into, through, and out of the pathway.
Figure 3. Schematic of three important types of steps in metabolic pathways that are key targets of regulation.
Attribution: Original work - Marc T. Facciotti
When to regulate?
A cell is always regulating the flow of molecules through its metabolic pathways. Understanding when to change the regulation of different metabolic pathways is the critical issue. When does the organism start to run glycolysis? When does it ramp up nucleotide biosynthesis and turn down amino acid biosynthesis? The short answer is that pathways are up-regulated when they are needed and down-regulated when they aren’t. But how does the organism know when something is needed and something else isn’t? The answer to this question is a bit more involved than what we can discuss in the time and space provided here. However, we can intuit that whatever these processes are, they must involve the sensing of environmental and/or cellular information. Something must be measured.
What to measure?
In the context of metabolic pathways, what information could be measured to help a cell make decisions about whether to up- or down-regulate flow through a metabolic pathway? Since metabolic pathways are in “the business” of consuming input molecules and converting them into some other product, it might be reasonable to expect that knowing whether there is enough product in the cell already (in which case the pathway doesn’t need to run) or whether there is enough of the original substrate around to feed the pathway could be useful information to know for regulation. Other cellular information of relevance to metabolism that might be useful to know about when deciding on whether to up- or down-regulate a pathway is the general level of usable energy (e.g. the balance between levels of ATP, ADP, and AMP) and the availability of reduced and oxidized electron carriers (e.g. the balance between NADH and NAD+). Clearly, in some cases the levels and balance between other molecules will also be important.
How can the cell use molecular information to make metabolic decisions?
Above we suggested some rationale about the regulation of metabolic pathways. We proposed that metabolic pathways must be regulated to manage cellular resources (why) and that this regulation should happen in response to changing cellular needs (when). We propose that regulation happens when branch points, irreversible reactions, and rate-limiting steps are found (where). Furthermore, we suggest that regulation happen by controlling the abundance and/or activity of the enzymes that catalyze reactions in a pathway (who). Finally, we posit that knowing the abundance of pathway inputs and products as well as general indicators of cellular energy and redox stores (what) is generally useful for cellular decision-making.
This leaves us to answer the final question. How? How can a cell use abundance of cellular molecules to inform enzymes catalyzing key steps in metabolic pathways about whether they are needed more or less when cellular conditions change? The complete answer to this question is, like seemingly everything else in biology, multifaceted, involving different targets of regulation (e.g. the genes encoding an enzyme to the enzyme itself) and different mechanisms. Here we briefly discuss one mechanism that links back to an earlier lesson on proteins; allosteric regulation.
Allosteric regulation involves the binding of molecules to allosteric sites on an enzyme that are by definition not in the active site. This binding alters the protein structure and can cause in different instances both either up or down regulation of enzyme activity. Often, enzymes that are the subject of allosteric regulation can be influenced by more than one ligand. By binding different phosphorylation states of ATP (i.e. AMP, ADP, and ATP) at allosteric sites enzymes can measure the ratios of ATP to its other forms and thus assess the energy “status” of the cell.
Examples of metabolic regulation by allosteric binding
Reactions with large negative ∆G
An early intermediate step of glycolysis, the phosphorylation of fructose-6-phosphate by ATP to yield fructose-1,6-bisphosphate and ADP, has an approximate ∆G of -19kJ/mol under cellular conditions. This is one of three largest, by comparison to all other reactions in the pathway, drops in free energy in the glycolytic pathway. In mammalian systems, the enzyme catalyzing this reaction, phosphofructokinase, is both positively and negatively regulated by multiple small molecules. ATP, citrate, and phosphoenolpyruvate (PEP) have been found to bind allosteric sites on the enzyme and lead to a lowering of enzyme activity. AMP, ADP, and fructose-2,6-bisphosphate (a product created by another enzyme) each increase phosphofructokinase activity. A number of other small molecules have also been found to influence enzyme activity in the test tube, but these are typically considered to play an insignificant role in the living system.
Given the central role of the glycolytic pathway in both energy harvesting and the creation of key precursors for other metabolic pathways, it is perhaps not surprising to have this enzyme’s activity influenced by a measure of the energy status of the cell (the ATP/AMP ratio) and by molecules in connected pathways (PEP and citrate). It is perhaps easiest to understand that when the enzyme senses low energy levels in the cell, when the ATP/AMP ratio is low, that the enzyme committing sugar to enter the energy extraction phase of glycolysis should become more active and that the converse should be true when ATP is abundant.
Figure 4. The enzyme phosphofructokinase is regulated allosterically both positively and negatively by numerous small molecules indicators of cellular energy state and of key concentrations of pathway intermediates.
Attribution: Original work - Marc T. Facciotti
An example of branch point regulation can be found in the pathways leading to the biosynthesis of aromatic amino acids (i.e. Phenylalanine, Tyrosine, and Tryptophan). The synthesis of each of these three amino acids begins with the production of the compound Chorismate. This compound can then be taken down two independent pathways. The first leads to Phenylalanine and Tyrosine biosynthesis while the second leads to Tryptophan biosynthesis. The enzyme catalyzing the first step in the path towards Phenylalanine and Tyrosine, Chrosimate mutase, is negatively regulated by the two products of the pathway and activated by Tryptophan, the product of the second pathway. Meanwhile, the enzyme Anthranilate Synthase is negatively regulated by the final product of its pathway, Tryptophan. The feedback of end-product levels on the enzymes responsible for catalyzing the synthesis of these three amino acids allow the cell to decide how to best utilize the stock of Chorismate and make that decision at the one of the core branch points for its use.
Figure 5: Regulation of branchpoint enzymes in the biosynthesis of aromatic amino acids. Dashed arrows indicate one or more steps are still required to create product of the pathway and these are not drawn explicitly. Note that this is also an example of pathway products feeding back to regulate upstream enzyme activity.
Attribution: Original work - Marc T. Facciotti
Shewanella are Gram negative, motile rods with positive oxidase and catalase reactions. Shewanella spp. are ubiquitous in natural environments, occurring mainly in marine environments, iced fish, proteinaceous foods, and occasionally clinical samples. The genus consists of more than 50 species that show various unique activities, such as metal reduction and trimethylamine production. Some of the species can be recovered from food and are regarded as fish spoilage bacteria some species grow at ≤4 °C and produce a variety of volatile sulfides, including H2S. In marine fish, they reduce trimethylamine oxide (TMAO) to trimethylamine (TMA), which has a fishy smell. These psychrotrophic Shewanella spp. are important in the food industry. This chapter describes the attributes of Shewanella, mainly focusing on food-related species. Here, the general characteristics and behavior in iced stored fish of, and off-odor compound production by, Shewanella spp. involved in food spoilage are described.
Colleges Reinvent Classes to Keep More Students in Science
DAVIS, Calif. — Hundreds of students fill the seats, but the lecture hall stays quiet enough for everyone to hear each cough and crumpling piece of paper. The instructor speaks from a podium for nearly the entire 80 minutes. Most students take notes. Some scan the Internet. A few doze.
In a nearby hall, an instructor, Catherine Uvarov, peppers students with questions and presses them to explain and expand on their answers. Every few minutes, she has them solve problems in small groups. Running up and down the aisles, she sticks a microphone in front of a startled face, looking for an answer. Students dare not nod off or show up without doing the reading.
Both are introductory chemistry classes at the University of California campus here in Davis, but they present a sharp contrast — the traditional and orderly but dull versus the experimental and engaging but noisy. Breaking from practices that many educators say have proved ineffectual, Dr. Uvarov’s class is part of an effort at a small but growing number of colleges to transform the way science is taught.
“We have not done a good job of teaching the intro courses or gateway courses in science and math,” said Hunter R. Rawlings III, president of the Association of American Universities and a former president of Cornell University and the University of Iowa. “Teaching freshman- and sophomore-level classes has not had a high enough priority, and that has to change.”
Multiple studies have shown that students fare better with a more active approach to learning, using some of the tools being adopted here at Davis, while in traditional classes, students often learn less than their teachers think.
The University of Colorado, a national leader in the overhaul of teaching science, tested thousands of students over several years, before and after they each took an introductory physics class, and reported in 2008 that students in transformed classes had improved their scores by about 50 percent more than those in traditional classes.
At the University of North Carolina, researchers reported recently that an overhaul of introductory biology classes had increased student performance over all and yielded a particularly beneficial effect for black students and those whose parents did not go to college.
Given the strength of the research findings, it seems that universities would be desperately trying to get into the act. They are not. The norm in college classes — especially big introductory science and math classes, which have high failure rates — remains a lecture by a faculty member, often duplicating what is in the assigned reading.
There are many explanations, educators say, including the low value placed on teaching, tradition, pride and the belief that science should be the province of a select few.
“What drives advancement at universities is publishing research and winning grants,” said Marc T. Facciotti, an associate professor who will teach a revamped biology course here in the winter quarter. “Teaching isn’t a very high priority.”
Noah Finkelstein, a physics professor and the director of Colorado’s overhaul efforts, added: “Faculty don’t like being told what to do, and there are people who push back and say they can figure it out on their own and they know what works for them. There’s plenty of data that says they’re mistaken.”
Employers and government officials have spent years complaining that there are too few people — and especially too few women and blacks — with degrees in math and science.
In fact, there is no shortage of interested students, but failure rates in the beginning classes are high. At four-year colleges, 28 percent of students set out as math, engineering and science majors, but only 16 percent of bachelor’s degrees are awarded in those fields. The attrition rate is highest among women and blacks.
“A lot of science faculty have seen themselves as gatekeepers,” said Marco Molinaro, an assistant vice provost here at Davis and director of its effort to overhaul science courses. The university has received grants from the Association of American Universities, the Bill & Melinda Gates Foundation and the Helmsley Charitable Trust.
Rather than try to help students who falter in introductory classes, he said, “they have seen it as their job to weed people out and limit access to upper-level courses.”
The project here borrows elements from many sources, including more than a decade of work at the University of Colorado and other institutions software from the Open Learning Initiative at Carnegie Mellon University Carl E. Wieman, a Nobel Prize-winning physicist at Stanford who founded Colorado’s project and a parallel effort at the University of British Columbia Eric Mazur, a Harvard physicist and author of the book “Peer Instruction” and Doug Lemov, a former teacher and author of “Teach Like a Champion.”
Many of the ideas — like new uses of technology, requiring students to work in groups and having them do exercises in class rather than just listen to the teacher — have caught on, to varying degrees, in grade schools and high schools. But higher education has been slower to change, especially in giant courses with hundreds of students.
While teachers at lower levels receive training in educational theory and teaching methods, most college instructors acquire none.
“Higher education has this assumption that if you know your subject, you can teach it, and it’s not true,” Dr. Uvarov said. “I see so much that I was missing before, and that was missing in my own education.”
Of course, telling experienced teachers that they need to learn how to teach does not always go over well, especially when they have tenure. So the project here began with graduate students who work as teaching assistants in biology and are required to have extensive training in teaching techniques. For an introductory science course, in addition to giant classes taught by faculty members, there are twice-weekly discussion sessions with two dozen students, led by teaching assistants.
“Unlike the profs, we could tell the T.A.s what to do,” said Christopher Pagliarulo, an associate director of Dr. Molinaro’s team.
The team tested students’ grasp of basic concepts before and after taking introductory classes, then it showed professors that their students were gaining much less than they had thought — results that convinced some professors of the need for change.
“There’s some ego involved, and it’s hard to hear that what you’ve been doing doesn’t necessarily work,” said Mitch Singer, the first professor on the Davis campus to teach a new-style introductory biology class, which is underway this quarter after months of preparation. “I think it’s also dawned on some professors that their T.A.s are now better teachers than they are.”
Faculty members say some colleagues are reluctant to jettison established lesson plans and accept a more unpredictable, boisterous classroom that puts students at center stage and forces professors to adapt. “It’s more work, and you’re not as in control,” Dr. Singer said.
The transition here has barely started — only the biology teaching assistants, plus a few faculty members in biology and chemistry, have undergone any retraining — but already the differences are plain. In their classes, Dr. Singer and Dr. Uvarov walk up to students, pace the aisles, and eavesdrop on working groups. They avoid simple yes-or-no questions and every query has a follow-up, or two or three.
Before each biology discussion session, students are supposed to go online to do some reading and answer questions. The teaching assistants then know who has done the reading, who has understood it and whether the group is weak in some spots, so they can tailor lessons accordingly. Students complain about being unable to escape scrutiny, but they acknowledge that they learn more. “I don’t like getting called on like that,” said Jasmine Do, a first-year student who was one of those singled out by Dr. Uvarov. “But it makes you participate and pay attention because there’s always something new going on, and it makes the time go by really fast.”
Faculty members have smartphone apps that let them call on students at random, rather than just on those who volunteer. When the instructors post multiple-choice questions on big screens, students answer with remote controls, providing instant feedback on how much information is sinking in and allowing faculty members to track each student’s attendance and participation, even in a class of 500.
“It’s already like night and day,” Dr. Singer said. “In a few years, it’ll be like day in the summer and night in the winter.”