CHM333 LECTURES 4 & 5: 8/31 9/2/07 FALL 2009 Professor Christine Hrycyna HOW ARE AMINO ACIDS MADE?

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1 HOW ARE AMINO ACIDS MADE? - Many organisms can make all 20 of the amino acids o Bacteria, yeast, and plants - Some amino acids are made from common metabolic intermediates directly o For example, alanine is made from pyruvate (transamination of pyruvate with glutamate as the amino donor) - Some amino acids are made as products from long and complex pathways o For example, aromatic amino acids are made from the shikimic acid pathway - Humans and other animals CANNOT make some of the 20 amino acids o These are termed ESSENTIAL AMINO ACIDS Arginine and Histidine are essential only in babies or in people with extreme metabolic stress disease Conditional Essential Amino Acids - Foods vary in protein quality o Content of essential amino acids - Animal proteins are often of a higher quality than vegetable proteins o Cereals are deficient in Lys o Legumes are low in Met and Cys o So vegetarians need variety 17

2 - Since human cannot make all 20 aa s we are susceptible to protein malnutrition especially in children and elderly adults - Disorder called Kwashiorkor Protein disorder in children Prevalent in overpopulated areas, particularly sections of Africa, Central & South America, and South Asia Areas of famine, limited food supply, diet mainly consisting of starchy vegetables Diet is adequate in calories (energy) but deficient in amino acids Symptoms include: lethargy, irritability, protruding belly, changes in skin pigment, hair changes, increased infections due to damaged immune system, enlarged liver, renal problems. Insulin levels stay high, subcutaneous fat store retained, skeletal muscle protein not available to visceral organs Onset often precipitated by increased demand for protein (infection, metabolic stress). Growing kids with poor diet = infection, hospitalized adults = acute stress. Little muscle wasting or fat depletion ; not noticed Ultimately leads to shock, coma and death Treatment: Depends on severity Increase calories from proteins usually in form of dry milk If not treated soon enough, permanent physical and intellectual disabilities can result HOW CAN WE EXPLOIT THE FACT THAT WE HAVE ESSENTIAL AMINO ACIDS? 18

3 EXAMPLE: Use in agriculture: - Pathways leading to essential amino acids are good targets for herbicides - Glyphosate (RoundUp) (N-phosphonomethyl-glycine) o Inhibitor of EPSP synthase, an enzyme in the shikimic acid pathway o Blocks the production of aromatic amino acids o Non-selective killer of anything green! - Glyphosate is relatively non-toxic to humans because we do NOT make our own aromatic amino acids. - RoundUp Ready soybeans and cotton carry an altered gene for EPSP synthase introduced into the plants using biotechnology - Altered gene encodes a protein that resists the RoundUp inhibitor 19

4 Noncovalent Interactions: Relatively weak, reversible, specific 1. van der Waals forces 2. Ionic Bonds 3. Hydrophobic Interactions 4. Hydrogen Bonds (electronegative atom (e.g. O or N) interacts with H atom that is partially positive (i.e. attached to N, O or S) BUFFERS - Buffers are extremely important in biological systems - Buffers are solutions that resist changes in ph upon addition of acid or base - Examples: o Maintaining blood ph o Maintaining physiological ph inside cells o More in a bit, but first let s review the basics of acid/base chemistry - WATER is the solvent of choice for biological systems o Constitutes 70-85% of cell weight, typically o Important as a solvent and a reactant in biochemical reactions o Helps regulate temperature since it is able to absorb large amounts of heat o Helps regulate intracellular ph o Used for transport delivers nutrients and removes waste from cells o Water is a unique solvent whose properties are extremely important to biochemistry. 20

5 CHM333 LECTURES 4 & 5: 8/31 9/2/07 FALL 2009 Professor Christine Hrycyna - In water, the hydrogen atoms have a partial positive charge, and the oxygen atoms have a partial negative charge - Water is a dipole because of its geometry and the difference in electronegativity between hydrogen and oxygen. Oxygen is more electronegative than hydrogen - The polar nature and geometry of the water molecule allows water molecules to form hydrogen bonds with each other and with dissolved hydrophilic substances. - Hydrogen bonds between water molecules= electrostatic attraction between the oxygen atom of one water and the hydrogen of another - Water can also form hydrogen bonds with functional groups of hydrophilic (polar or ionic) biomolecules and organic compounds. hydrogen bond donors hydrogen bond acceptors 21

6 - Hydrogen bonds also can form between two biomolecules (e.g. proteins & DNA) - The 3-dimensional structure of many biological molecules (eg. proteins) and macromolecular structures (eg. membranes, DNA) is determined by hydrogen bonding and hydrophobic interactions - Hydrogen bonds are weak but their abundance makes them important! REACTIONS OF WATER: - Reversible self-dissociation = ionization o Generates H + and OH - o Can be described by the following equilibrium: *Note hydrogen atoms do not exist as free H + in solution. Actually are hydronium ions (H 3 O + ). For simplicity, we just write H +. - Express extent of ionization quantitatively: Use law of mass action to define the equilibrium point of the dissociation reaction: K eq is defined as the ratio of the concentrations of the products and reactants. Units used to define concentration are Molarity (M) = moles/l - K eq for pure water determined experimentally to be 1.8 x M at 25ºC - Concentration of pure H 2 O = 55.5M (weight of water in 1 L (1000 g) divided by mw of 18) (M 2 ) (under square root should read M 2 ) - This is the basis of the ph scale! - These numbers are very small and difficult to work with, so in 1909 Soren Sorenson introduced the term ph to more conveniently express [H + ]. - Defined ph as the negative logarithm of the hydrogen ion concentration: - Also the same as log 1/[H + ] - p is an operator means to take the negative log of - Example: poh = -log[oh - ]; ph of pure water? [H + ] = 1 x 10-7, ph = -log (1 x 10-7 ) = 7 22

7 - Back to water ionization: - Take log of both sides for convenience: log (1 x M 2 ) = -log [H + ] + -log [OH - ] 14 = ph + poh - The ph scale ranges from 0 to 14 - ph scale is LOG BASED! Used to keep track of large changes important to acids and bases - Defined ph as the negative logarithm of the hydrogen ion concentration: - Water ionization: - Take log of both sides for convenience: log (1 x M 2 ) = -log [H + ] + -log [OH - ] 14 = ph + poh - The ph scale ranges from 0 to 14 - ph scale is LOG BASED! Used to keep track of large changes important to acids and bases - Important to remember that the scale is exponential o One ph unit = 10 times more acidic or basic 23

8 Remember: Add acid, ph gets smaller Add base. ph gets larger - Applies to other acids and bases, not just water o Acid = Releases proton in water (proton donor) o Base = Accepts proton in water (proton acceptor) o Water can act as an acid and a base = amphiprotic o Strength of an acid is defined as its tendency to release a proton (dissociate) - Define dissociation for an acid: (Conjugate base) - Conjugate Base: base formed by the removal of a proton from an acid - The acid and conjugate base are complementary species every acid has a conjugate base - Should be able to identify acids and their conjugate bases: Acetic acid/acetate pair: - Just as for water, we can write an equilibrium constant for the dissociation of the acid (K a ) - We want K a in convenient terms. Apply p rule and take the negative log of K a to get pk a. - pka is a QUANTITATIVE measure of acid strength. o Smaller pk a Stronger acid o Larger pk a Weaker acid (stronger base) o Opposite of Ka where a LARGE number indicates strong acid o Large Ka means mostly dissociated into H + and A-, not much HA left. Numerator large, denominator small large number K a large = pk a small 24

9 - Examples of acids: o Strong acids HCl and HNO 3 Dissociate completely in water [H + ] concentration is approx. equal to the [acid] in solution Ka is very large; pka is small - Weak Acids: o Common in biological systems will focus on these o Acetic acid, phosphoric acid, carbonic acid, and lactic acid are common o Not completely dissociated in water, so [H + ] will be much lower that [HA] o Need to be concerned with equilibrium in solution o Amino acids are also weak acids o pk a >1 25

10 - Henderson-Hasselbalch Equation o Equation that describes the behavior of weak acids in solution o Allows us to calculate the concentration of an acid and conjugate base at various ph - We measure ph - Since the ph of a solution is determined by the relative concentrations of acids and bases, let s express this in terms of ph. - Isolate [H + ] factor - Take log of both sides - Rearrange to get log[h + ] by itself - We then apply the definitions of ph and pka to get: [conjugate base] [acid] USE THIS RATIO OF THE TWO SPECIES TO CALCULATE BUFFER COMPONENTS! - Note that if [A - ] = [HA], the following is true: ph = pka + log1 (log 1 = 0) ph = pka MAXIMUM BUFFER CAPACITY **A buffer is effective at ph +/- 1 of the pka** This is mathematical proof of the behavior of buffers. At or near the point where the conjugate base and acid concentrations are equal is the best region of buffering. 26

11 If we look at a titration curve for a weak acid such as acetic acid, you can see the behavior. - Plot equivalents of OH - vs. ph - To start, acid form predominates fully protonated - As NaOH is added gradually, OH - combines with free H + in solution to form H 2 O. - As free H + is removed, CH 3 COOH dissociates further to stay in equilibrium. - Net result More and more CH 3 COOH ionizes, forming CH 3 COO - - Equivalence point is reached around ph 7 when all CH 3 COOH has lost protons - One titratable proton from CH 3 COOH is titrated (removed) - Midpoint of curve (inflection point) is where there are equal amounts of acid and conjugate base. The ph of this solution at the midpoint is the pka! REMEMBER: - Titration curves for weak acids show that a weak acids and conjugate base can act as a buffer. Resists change in ph upon addition of acid or base. - Buffers best around the pka (just like equation demonstrated!) - Established an equilibrium between buffer components (remember log of the ratio!) large additions needed to make changes in ph - The H-H equation represents removal of one proton from an acid one equilibrium between acid and conjugate base. 27

12 - Some buffers are POLYPROTIC that is they have more than ONE acidic proton - All H + do NOT dissociate at the same pka but are released SEQUENTIALLY at different pka s start at lowest ph and go to higher ph For example, phosphoric acid has 3 titratable protons: - 3 Different equilibria exist - Note the conjugate base from 1 st ionization is the acid for the 2 nd and so on - Each can be represented by the H-H equation - Write a separate equation for each step - To use H-H: select the reaction and pka closest to the ph of interest AN EXAMPLE: 28

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