As the potential energy of a system is released (here converted to kinetic energy) the system becomes more stable and the released energy is available to create change (do “work”) within the system. Similar energy conversions govern cellular metabolism.
The Laws of thermodynamics govern energy flow in living systems the same as in the non-living world. Energy can’t be created nor destroyed, it only changes form; AND when it does there is always less usefull (ie. “free” ) energy left afterward
In living cells, some of the energy liberated in a “breakdown” or catabolic reaction can be saved in the form of ATP, but much is still lost as heat.
This slide was included in the Ch 7 presentation; lets look at what’s going on here in a bit more detail
Cells consume ATP at a phenomenal rate so it must constantly be regenerated using the energy supplied from the environment as sunlight or chemical bond energy
Catabolic (“breakdown”) reactions liberate chemical bond energy, some of which is captured and transferred to ATP via linking ADP and P. Anabolic (“build-up” or synthesis) reactions require an external source of chemical bond energy, which is usually supplied by the hydrolysis of ATP to ADP and P.
Some reactions are “spontaneous” - they go with the flow, while other reactions are not. A spontaneous reaction involves an energy change that is in keeping with the second law of thermodynamics which means that as the reaction proceeds energy is released and the system contains less high quality energy after the reaction than before. A nonspontaneous reaction works has the opposite properties and so requires a high quality energy input. Both are possible in cells, and both can be catalyzed by enzymes.
All chemical reactions involve some sort of free (ie. “usefull”) energy change G. Free energy is either released (left) or an input required (right) as the reaction progresses. If more is released, then the products contain less than the reactants (red rxn on left) and the free energy change G is negative (-). Such rxns, in effect, work in accordance with the second law of thermodynamics - they “go with the flow” so to speak, and are referred to as being “spontaneous.”
The (Gibbs) Free energy change that accompanies a reaction is a composite of the change in chemical bond energy or enthalpy, H, and the change in the product of temperature (in o K) times entropy, T S that a reaction produces. G = H - T S Josiah Willard Gibbs was a bachelor engineer who received his Ph.D. and did all of his subsequent work at Yale. He was reclusive genius who founded the field of chemical thermodynamics in he late 1800’s. The effort involved in comprehending his work is still driving students (not to mention professors) crazy to this very day. Gibb’s work received somewhat limited acclaim until 1923 when Lewis and Randall publishedThermodynamics and the Free Energy of Chemical Substances, which introduced the methods of Gibbs to chemists world-wide. (Wikipedia)
To confuse the issue further, we can express G in terms of the concentration of reactants and products at the equilibrium point. Where K eq for the reaction A+B -> C +D or is defined as:
An oversimplified (but less mind-bending for the symbolically challenged) way to look at this is that the equilibrium constant depends on the relative concentrations of the reactants (A and B) and products (C and D) at the point in the reaction where neither is changing any further. So a reaction’s K eq is calculated from measurable quantities, and therefore the subsequent calculation of G can be done based on actual measurements for any set of conditions. A key point here is that the K eq and G for a reaction is dependant only on the chemical nature of the reactants and products, their concentrations, and the conditions under which the reaction is carried out. The speed at which the reaction occurs is not relevant (it’s independent).
So, what’s all this have to do with enzymes? Hold that thought and consider the following analogy based on jumping beans (its not perfect, but few analogies are). Jumping beans are seeds of a Euphorb shrub, Sebastiania palmeri or S. pavoniana that have become infested with the larva of a moth, Cydia deshaisiana, these are found in the Mexican desert. The larva consume the seed contents and persist within the seeds even after they’ve fallen from the plant. Temperature changes cause the larvae to contract violently causing the whole seed to “jump.”
The beans will often “jump” at random times with random orientations (in any direction) and energies. So “beans” in a box will jump with an occasional jump being energetic (high) enough and oriented such that the bean clears the box walls
OK, so lets let the beans in the left hand box represent reactants and those in the right hand box represent products. I know, you can argue that, unlike real reactants and products, the beans are still the same regardless of which box they’re in, but let’s ignore that. Given enough time, some beans would manage to jump the barrier (wall) separating the two compartments. That is, “reactants” would become “products”.
As pictured, the “product” beans are unlikely to be able to jump back and become reactants again due to the difference in the depth of the compartments. This is analogous to a change (a decrease) in H between reactants and products that would be expected for a (exothermic) “spontaneous” reaction where G is negative and large (free energy is given off) HH
SS In some cases, both H and S ( both the height and width of the boxes) differ. A given # of molecules occupying a larger space = an increase in disorganization or entropy ( an increase in S). Such a reaction in which H decreases and S increases, would be strongly exergonic and spontaneous. The combustion of paper (combining O 2 and cellulose or linked glucose) to CO 2 and H 2 O for example.
So what’s all of this got to do with enzymes? The short answer to this point is, “nothing.” The G, H, and S of any reaction, as stated before, are functions of the chemical nature of the reactants and products and/or the conditions under which the reaction is carried out. Regardless of whether an enzyme is involved or not. An enzyme will make it possible for an energetically feasible reaction to occur faster, it won’t make a non-feasable reaction possible. Enzymes accelerate reactions; they can’t affect the extent to which a reaction goes to completion (the K eq ) or the G. This acceleration occurs via the lowering of the activation energy barrier.
Enzymes are biological catalysts that allow the chemical reactions of life to occur much faster than they could otherwise (with a much lower energy input). They are mostly catalytic proteins. Very specific (have specific substrates) Not consumed Activity can be modulated (controlled) They don’t alter the nature of the energy change that a given reaction entails, they merely make “possible” reactions go faster.
For the combustion of glucose Without an enzyme, the large activation energy requirement must be supplied by the application of an external source of heat. In cells, glucose is “combusted” in many small steps the combined G of which is the same as if it had all been accomplished in one step as above.
The key to reducing the needed activation energy is the holding of the reactants in the active site in a manner such that less activation energy is needed to break old bonds or make new ones.
Reactions that are non-spontaneous, such as synthesis reactions (more chemical bonds and order are being created), require an energy input to provide the necessary + G. This is usually accomplished by linking such reactions with the hydrolysis of ATP; the high energy P so produced is first linked to a substrate(s) to provide the needed + G.
To synthesize the amino acid glutamine from glutamic acid the glutamate is first phosphorylated via ATP hydrolysis.
Remember, Enzymes are: Highly substrate-specific. Affected by the relative concentrations of enzyme and substrate to the point where the reaction rate is saturable. Subject to competitive and non-competitive inhibitors. Subject to (affected by) the physical (temperature) and chemical (pH, [salt]) conditions under which they operate. Most often operate as part of a metabolic pathway where the product of one enzyme becomes the substrate for the next.
OK, lets look at how relative E & S concentrations effect what enzymes do in a closed system (a test tube). Enzymatic reactions take place in two steps, substrate binding (E+S) to form an ES complex followed by catalysis, the conversion of S to P. E + PES E+S The E+SES binding step has a variable speed that depends on [S] for a given[E] While theESE + P step has a speed that is fixed by the properties of the enzyme.
The rate of product, P, production increases as [S] (concentration of S) increases, but only to a limit.
For each [S], the reaction rate increases to some limit; the leveling off point for each [S] represents the equilibrium point for that [S]. You can see from this graph already that even though [S] is increasing in even increments, the increase in rate (line slope) is less for each successive [S] increase.
To show how [S] influences the reaction rate (speed of S converting to P) more clearly, it helps to plot a second graph of Velocity (rate) vs. [S]. To do this, we take data from the first graph (rate vs. time for each [S]) shown here and plot a second graph.
We need to select a standard time at which to compare rates - represented by the vertical line drawn here. Next note the slope of each [S] line at this point (the slope for each line expressed in moles/min); this can be calculated from absorption readings using the product absorption coefficient.
For each [S] the rate (tangent line slope at our selected T value) is plotted on a new graph of velocity (slope) vs. [S]
The new graph (on the right) when complete will look something like this.
This new graph now allows us to see other relationships, including that an enzymatic rxn can be saturated with S. Increasing [S] beyond that which gives the maximum rate or velocity, V max won’t increase the rxn velocity further. How about a simpler monkey-peanut analogy?
If you found that confusing, how about Monkeys and Peanuts? This analogy has been around for awhile in various books, etc. I stole these illustrations from: http://www.creativebiology.co.uk/#/enzyme-monkey-nuts/4546595080 Lets assume that we have some monkeys and lots of peanuts, and that we know the average time it takes for a monkey to shell a peanut and pop it into its mouth.
So a given mix of [E] (Monkeys) and [S] (substrate) would look like this.
Lets keep [E] (Monkeys) constant and increase [S] (peanuts). How would this affect the time it takes to go from E+S to ES? What about the time it takes to go from ES to E + P? If we added more peanuts still, at some point the monkeys would have such easy access to the peanuts that adding even more yet would not allow them to work any faster (E+S -> ES) and we’d be at the saturation point = V max
To reiterate: At low [S] the E (monkeys), take a while to find the few S molecules (peanuts) available in the test tube (their cage).
As [S] (the number of peanuts) is increased, the E (monkeys), find it easier to find S molecules (peanuts) in the test tube (their cage) and the rate of S-to-P conversion increases.
As [S] (the number of peanuts) continues to increase (because we are making more available) at some point, the enzyme molecules (monkeys) simply can’t bind them any faster and the system is saturated. Any further increase in [S] will not increase the rate of product formation which is already at V max
One additional piece of info: the “k m value” of an enzyme is specific to each substrate that it can work with. For example our turnip peroxidase can work with a number of materials as the non-peroxide substrate which is why the letter “R” is used to designate it: RH 2 + H 2 O 2 R + 2 H 2 O
Some specific K m s for comparison: A few other “Rs,” other substrates that peroxidase can also work with, and their Km values for comparison: Substrate K m * 3,3’,5,5’-tetramethylbenzidine0.045 Guaiacol0.14 2,2’-azino-bis(3-ethylbenzthiazol-6-sulfonic acid0.19 Phenol1.17 O-Cresol16.7 * K m values are mMoles of substance giving ½ Vmax under standard conditions
K m is the [S] that produces 1/2 V max so its reasonable that smaller K m values indicate a tighter binding of the substrate (and the reaction rate will be faster as a result on an equimolar basis). Different materials (Rs) will have a higher affinity for the enzyme = will bind better (= lower K m ), or a lower affinity for the enzyme = looser fit (= higher K m value). We chose guaiacol for our “R” compound because when it reacts it produced a colored product. The relative k m value was not an important consideration for our purposes.
The Michaelis-Menton equation can be derived from these values as shown; it is useful for determining how an enzyme will behave under different conditions, but its use will not be pursued further here. So IGNORE THIS for the exam, it’s just an additional comment
Remember, these aspects of enzyme kinetics are demonstrable in a closed system only - where product cannot be removed - with an enzymatically catalyzed reaction taking place in a test tube, such as we did in lab for example. In a cell, it’s a different ballgame as P is never allowed to build up. The effects of E & S concentration still apply however.
In a cell, enzymes are arranged into metabolic pathways where the product of one becomes the substrate for the next. A B C D E F Enz.1 Enz.2 Enz.3 Enz.4 Enz.5 Because products are removed as fast as they are made, the materials in pathway “flow” in the forward direction. Equilibrium, where the changes (and the accompanying energy changes)effectively come to a standstill DOSE NOT OCCUR, if it did it would = cellular death. Pathways can be linear as shown (see glycolysis) or cyclic, where F, with addition of another input, resynthesizes A and the the pathway cycles (see the citric acid cycle).
In living cells, the product of one enzymatic reaction becomes the substrate for the next.
To reiterate: At equilibrium G = 0; in living cells, reactions always proceed in the forward direction because in a metabolic pathway products are never allowed to accumulate, so the reverse reaction never occurs (therefore no equilibrium point is reached). In cellular metabolism an equilibrium state (no energy change going on) = death. Also, a reaction, such as glucose combustion, that is carried out in many small steps in living cells, still has the same cumulative overall G (on an equal volume basis) as it would if you simply burned a spoonful of it in an open flame.
BRANCHING PATHWAY: LINEAR PATHWAY:CYCLIC PATHWAY: ABCDE F KJI G NML H Metabolic pathways can take several forms
Intermediary metabolism can be defined as the sum total of all of the enzymatic pathways that are necessary for a cell to be able to sustain itself. The pathways of glycolysis, the transition reaction, and the TCA or Kreb’s cycle play central “clearing house” roles in intermediary metabolism in addition to being part of aerobic respiration which supplies ATP to the cell.
Enzymes can be inhibited (slowed down) by competitive (substrate-like) or non-competitive (enzyme shape- altering) materials. A key to understanding how these processes work is realizing that the binding between an enzyme and it’s substrate, or an inhibitor, is seldom “tight” so it’s an on again / off again affair that is affected greatly by (relative) substrate and inhibitor concentrations. This is especially true for competitive inhibition
Figure 5.10 A competitive inhibitor has at least part of it’s structure that is very similar in size and shape to the size & shape of the normal substrate. So the more Inhibitor floating around, the harder it is for the enzyme to find & bind to the substrate and product production (the reaction rate) slows down. Adding more substrate (changing the I/S ratio in favor of S) will speed things up again. Non-competitive Is work by changing the overall enzyme shape, including the shape of the active site, but because they bind at a separate allosteric site, adding more S has no effect (won’t speed things up again).
In some cases, substrate binding (in one subunit) can (allosterically) enhance the ease of substrate binding by others.
Temperature and solution ion concentration can also affect activity by affecting enzyme (and active site) 3D shape. The shape of the enzyme and its active site are affected by anything that alters all of the weak interactions that determine protein 3D shape. There is some concentration of ions, including H + (ie. pH) that allows the enzyme and active site to have their optimal shape. Under such optimal conditions, the enzyme works the best. Temperature also affects the rate molecules move around and bump into each other. So as temp. goes up, so does reaction rate until the point where denaturation sets in.