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		Quiz Me!	Energetics and Enzymes
		Lecture Index
		Course Index

Last revised: Saturday, September 25, 1999
Reading: Ch. 6 in text

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 1999. Thomas M. Terry

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A. The Logic of Cell Chemistry--an overview

• Think of cells in your body as factory:
  1. Day 1: eat only potato chips and OJ --> new human hair, muscle
  2. Day 2: eat only hamburgers & milk --> new human hair, muscle
  3. Day 3: eat only rice & beans --> new human hair, muscle
• You transform different chemicals into new human chemicals. HOW?

How can you build a complex chemical factory?

Requirements of the problem

• factory must be able to take any one of dozens of chemicals, transform them into thousands of different products
• factory must be able to quickly "tool up" to new products as demand changes, shut down unneeded products
• factory must be able to compete efficiently with other factories
• factory must be able to sense what is going on in world, respond to changes: grow, relocate, lay off workers, expand, set up new factories
• factory must be able to be miniaturized down to pinhead size
• factory must be totally autonomous: no outside help

Solution 1: pipes, valves, computers

• typical human engineering solution
• move chemicals around with pipes; control by valves; regulate by computer program
• extremely complex, elaborate, incapable of being miniaturized, replicated

Solution 2: broadcast, collision, specific reactors

• Cell uses a totally different solution to problem of being a chemical factory
• "Broadcast" metaphor: all reactants, products are broadcast into a common space (in higher cells several different spaces); analogous to radio waves all broadcast into one room
• Chemicals interact by random collisions. Total space must be small to allow all possible collisions
• Most collisions fruitless (like radio not being tuned in). Occasional collisions produce temporary association with a "specific reactor", like a radio locking onto a specific signal
• Fruitful collisions produce chemical change. Total = Metabolism

The "specific reactors" are Enzymes (protein)

• A major challenge of biology is to understand how enzymes work
• To understand enzymes, must first understand principles that govern chemical change:
  1. Energy
  2. Equilibrium
  3. Rate

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B. Energetics

Definition and Measures of Energy

• Energy = capacity to do work.
• Examples:
  
  • Light
  • heat
  • electricity
  • gravitational position
  • motion
  • chemical bonds
  • Energy
• Energy is measured in calories or joules.
• 1 calorie = 4.1840 joules. Physicists & chemists use joules; biologists typically use calories.
• calorie = energy to raise 1 gm water by 1^o C.
• Kcal = 1000 cal

Laws of Thermodynamics

• Thermodynamics = laws governing energy transfer.
• Originally from study of heat; later shown to apply universally to all forms of energy transfer
  1. 1st Law: in closed system, energy can neither be created nor destroyed, only changed in form.
  2. 2nd Law: energy transformations inevitably involve increased disorder, or entropy.
• Implications of 2nd Law:
  1. disorder is increasing
  2. processes go to equilibrium
  3. heat flows from hot to cold
  4. diffusion leads to substances becoming uniformly dispersed
  5. systems far from equilibrium acn do useful work; not possible after equilibrium
  6. is attained
  7. Entropy governs availability of energy for useful work.
• Note: biological systems are never at equilibrium (unless dead).

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C. The Nature of Chemical Change

All systems spontaneously move towards some equilibrium state

1. In principle, any chemical reaction is reversible.
2. Example: H₂ + O₂ --> H₂O. (explosive). But also to some extent H₂O -> H₂ + O₂
3. equilibrium constant K_eq = [products]/[reactants]
4. If K_eq is high (eg 100), reaction goes greatly to products; if low, goes towards reactants

Exact balance of product & reactant depends on free energies

• all reactions proceed in direction analogous to moving downhill, not uphill. "Downhill" = decreasing free energy. How to measure free energy in chemical terms?
• Gibbs Free Energy , or G, measures amount of energy involved in reaction ; is defined in terms of both heat and entropy:
• Definition: G = H - TS
  • whereG = change in free energy, a measure of useful work
  • H = change in heat content
  • S = change in entropy
  • T = absolute temperature in degrees Kelvin
• Example: in burning glucose (e.g. paper), H = -673 kcal, TS = - 13 kcal; total G = -686 kcal
• Cannot measure G exactly; but can measure G. In practice, use combination of tables (under standard contions of 1 molar concentration, pH 7, can calculate Go') and experiment (typically measure heat change, temperature)
• Conventions:
  • exergonic rxn has G <0 (e.g. -3 kcal, -15 kcal); system can do useful work, will occur spontaneously;
  • endergonic rxn has G >0 (e.g. +5 kcal, + 20 kcal); reaction will not occur spontaneously, can only occur if coupled to some other reaction that liberates even more energy

G is related to equilibrium

1. consider rxn. A + B <=====> C + D
2. Any reaction has some equilibrium state; given enough time to reach equilibrium, concentrations will remain constant
3. Keq = [products]/[reactants] = [C][D]/[A][B]

                     Keq    Go'	      comments
C, D = A, B	       1        0         no useful work
C, D = 10 x A, B      10     - 1.36      useful work
C, D = 100 x A, B     100    - 2.73      more useful work
C, D = 1000 x A, B    1000    -4.10     even more useful work

Rate of a chemical reaction is not predictable from G

• Compare piece of newspaper on windowsill with paper in fireplace. One browns over years, one in seconds.
• Same reaction in both cases: glucose + oxygen --> water + CO₂.
• Both reactions have exactly the same G. Difference is only in rate.
• You cannot predict how fast something will happen by knowing its G; what you can predict is which way the reaction will proceed, and how much energy will be released or consumed.

Rates are limited by Activation Energy

1. reacting molecules must have enough energy
   • rate can be increased by increased temperature, increases kinetic energy
2. reacting molecules must be in precisely correct relative orientation to form the transition state
   • chemical reactions involve rearrangements of electrons. Like going from one depression to another--even though second is more stable, first has its own stability, won't immediately change
   • this is extremely improbable, because requirements to form a new bond are very strict
   • even a fraction of an Angstrom out of alignment will reduce occurrence to negligible levels
   • when other molecular participants involved (e.g. acids, bases), extremely low probability that reaction will proceed at all
   • this low probability is defined by entropy.
3. Rates can be accelerated by catalysts
   • catalyst is anything that speeds up a reaction, but does not become part of the process
   • industry typically uses things like finely ground platinum, magnesium to speed up a number of rxns. These are non-specific catyalysts.

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D. Enzymes

Enzymes are named by adding the suffix -ase

• Example 1: the enzyme deoxyribonuclease, or DNAse, breaks down DNA
• Example 2: the enzyme lactase attacks the disaccharide sugar lactose

Enymes are three-dimensional stereospecific catalysts

1. Enzymes are typically large proteins, contain an active site
2. Substrate binds to active site in highly 3-D specific orientation.
3. Enzyme + Substrate form temporary chemical bonds, both weak and strong.
4. Enzyme facilitates bringing substrates into exact alignment needed for transition state to be achieved. This often involves temporary changes in shape of enzyme, called Induced Fit.
5. Enzyme dissociates from product after reaction is complete (thousandths of a second typically)
6. View animation of enzyme-substrate interaction
7. Enzyme is unchanged, able to recycle again
8. Typical equation: E + S ----> E-S complex ----> E + P

• Explore Ribonuclease (RNase) as a sample enzyme
• RNase is an enzyme that hydrolyzes (breaks down) RNA molecules. RNase is very common; it is found in saliva, in digestive fluids, even in body secretions. One reason for its ubiquity may be that many viruses contain RNA as their genetic material; failure to destroy extracellular RNA could mean more frequent infection by RNA viruses!
• RNse is a small enzyme, a single polypeptide chain with 124 amino acids.
• View static image showing 3^o structure in Ribonuclease enzyme
  
• Explore 3-D structure of Ribonuclease.. Click "Submit Request" to obtain dynamic image. Note: the free CHIME plug-in must be installed for this activity. Once the image loads, proceed as follows:
• For more information, including information on how to obtain CHIME and tutorials on how to use it, visit the Chime Resources at UMass page.)
  1. You should see a wireframe view of the RNase molecule on your screen, as follows:
     
     
  2. Hold down the mouse button (Macintosh) or click the right mouse button (Windows); a pop-up menu will appear. Select "Display", and then "Cartoons". You should now see a folded white ribbon (representing the protein) and a small yellow ribbon (part of the RNA substrate for the enzyme).
  3. Again use the pop-menu; this time select "Color" and then "Chain". You should see a blue ribbon (representing one of the structures, the polypeptide) and a green chain (a different structure, the RNA substrate).
  4. Try dragging your mouse over the object to make it rotate in space. Examine the three-dimensional relationship between the substrate and the enzyme. Note: Mac users, start dragging immediately after you click the mouse; if the pop-up menu appears, you waited too long to drag. To resize the image, hold down the shift key while you drag across the screen. Dragging downwards increases size, upwards decreases size.
  5. To see how the enzyme and substrate fit together, you must change the view option to see all atoms, not just the ribbon backbone. Use the pop-up menu to select "Display", "SpaceFill", and "Van der Waals Radii". Rotate and examine the relationship between enzyme and substrate. What shape is the active site? How tightly do the enzyme and substrate fit? The red balls represent water molecules. Your RNase molecule should now look like this:
     
     

View Enzyme Figures

Enzymes accelerate chemical reactions by factors of 10¹⁰ to 10¹⁵

1. Difference between presence or absence of enyzme is enormous.
2. Example: breakdown of urea (nitrogenous waste in urine) would take about a year without enzyme, only millionth of a second with enzyme.
3. Enzymes characterized by a turnover number: typically 1000s to millions of reactions per second
4. Cell chemistry is absolutely dependent on enzyme; if one type of enzyme if lost, that reaction will no longer occur at useful rates, for practial purposes will not occur at all.

Specificity of enzymes is variable

1. some enzymes work on one unique substrate only
2. others will accept a variety of substrates that have certain types of chemical similarity; will work better on some, poorer on others

Many enzymes require cofactors

1. some enzymes have tightly bound helpers called coenzymes or cofactors
2. Cofactors can be single metal ions (Mg, Zn, Co, Mn, etc)
3. Cofactors can be small organic molecule called coenzyme

Enzyme activity can be regulated in different ways

1. Consider Regulatory problem of cell: thousands of enzymes, each with a "mind of its own". Yet cell needs overall stability.
2. Example: synthesis of a certain amino acid. Reaction scheme looks like this:

   

3. Suppose supply of E in cell increases (e.g. eat a meal rich in E). How to shut down synthesis of E?
4. Cell's answer: Enzyme 1 is reversibly inhibited by E. Note that E is not the substrate, and chemically so different that it cannot bind to active site. How does E shut down Enzyme 1?
5. Enz 1 is a special type of enzyme called an allosteric enzyme. It causes feedback inhibition. Allosteric enzymes contains two distinct subunits, one with active site (binds substrate A and catalyzes reaction), one with allosteric site (binds E).
6. When E binds, causes shape change in the enzyme, this is transmitted to block activity of active site.
7. View animation of allosteric enzyme (by Dr. Steven Berg, Winona State University)
8. Net result: whole pathway is turned on or off as a unit by end-product. Called Feedback inhibition. Crucial to cell regulation.

View Enzyme Figures

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