Andy Howard Introductory Biochemistry Fall 2013, IIT Amino Acids Andy Howard Introductory Biochemistry Fall 2013, IIT

Amino acids and what they do We’ll show you all 20 ribosomally encoded amino acids and discuss their reactivities 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins Plans Amino acid properties Amino Acid Structures Chirality Acid-base behavior Abbreviations Side-chain reactivity Peptide bonds Peptides Proteins 08/29/2013 Amino Acids and Proteins

Let’s begin, chemically! Amino acids are important on their own and as building blocks We need to start somewhere: Proteins are made up of amino acids Free amino acids and peptides play significant roles in cells, even though their resting concentrations are low We’ll build from small to large 08/29/2011 Thermo II; Amino Acids I

Acid-Base Equilibrium In aqueous solution, the concentration of hydronium and hydroxide ions is nonzero Define: pH  -log10[H+] pOH  -log10[OH-] Product [H+][OH-] = 10-14 M2 (+/-) fact So pH + pOH = 14 derived formula Neutral pH: [H+] = [OH-] = 10-7M: pH = pOH = 7. 08/29/2011 Thermo II; Amino Acids I

So what’s the equilibrium constant for this reaction? Note that the equation is H2O  H+ + OH- Therefore keq = [H+][OH-] / [H2O] But we just said that [H+] = [OH-] = 10-7M We also know that [H2O] = 55.5M (= (1000 g / L )/(18 g/mole)) So keq = (10-7M)2/55.5M = 1.8 * 10-16M 08/29/2011 Thermo II; Amino Acids I

Henderson-Hasselbalch Equation If ionizable solutes are present, their ionization will depend on pH Assume a weak acid HA  H+ + A- such that the ionization equilibrium constant is Ka = [A-][H+] / [HA] Define pKa  -log10Ka Then pH = pKa + log10([A-]/[HA]) 08/29/2011 Thermo II; Amino Acids I

The Derivation is Trivial! Ho hum: Ka = [A-][H+]/[HA] pKa = -log10([A-][H+]/[HA]) = -log10([A-]/[HA]) - log10([H+]) = -log10([A-]/[HA]) + pH Therefore pH = pKa + log10([A-]/[HA]) Often written pH = pKa + log([base]/[acid]) 08/29/2011 Thermo II; Amino Acids I

Thermo II; Amino Acids I How do we use this? Often we’re interested in calculating [base]/[acid] for a dilute solute Clearly if we can calculate log([base]/[acid]) = pH - pKa then you can determine [base]/[acid] = 10(pH - pKa) A lot of amino acid properties are expressed in these terms It’s relevant to other biological acids and bases too, like lactate and oleate 08/29/2011 Thermo II; Amino Acids I

Reading recommendations If the material on ionization of weak acids isn’t pure review for you, I strongly encourage you to the relevant section of Garrett & Grisham We won’t go over this material in detail in class because it should be review, but you do need to know it! 08/29/2011 Thermo II; Amino Acids I

pKa values for weak acids Acid Base pKa Acid Base pKa Form Form Form Form Formic acid Formate 3.8 Acetic acid Acetate 4.8 Lactic acid Lactate 3.9 H3PO4 H2PO4- 2.2 H2PO4- HPO4-2 7.2 HPO4-2 PO4-3 12.7 H2CO3 HCO3- 10.2 NH4+ NH3 9.2 CH3NH3+ CH3NH2 10.7 Alanine+ Alanine0 2.4 Alanine0 Alanine- 9.9 08/29/2011 Thermo II; Amino Acids I

So: let’s look at amino acids The building blocks of proteins are of the form H3N+-CHR-COO-; these are -amino acids. But there are others, e.g. beta-alanine: H3N+-CH2-CH2-COO- 08/29/2011 Thermo II; Amino Acids I

Thermo II; Amino Acids I These are zwitterions Over a broad range of pH: the amino end is protonated and is therefore positively charged the carboxyl end is not protonated and is therefore negatively charged Therefore both ends are charged Free -amino acids are therefore highly soluble, even if the side chain is apolar 08/29/2011 Thermo II; Amino Acids I

Thermo II; Amino Acids I At low and high pH: At low pH, the carboxyl end is protonated At high pH, the amino end is deprotonated These are molecules with net charges 08/29/2011 Thermo II; Amino Acids I

Identities of the R groups Nineteen of the twenty ribosomally encoded amino acids fit this form The only variation is in the identity of the R group (the side chain extending off the alpha carbon) Complexity ranging from glycine (R=H) to tryptophan (R=-CH2-indole) Note that we sometimes care about -amino acids that aren’t ribosomal—like ornithine ornithine 08/29/2011 Thermo II; Amino Acids I

Let’s learn the ribosomal amino acids. We’ll walk through the list of 20, one or two at a time We’ll begin with proline because it’s weird Then we’ll go through them sequentially You do need to memorize these, both actively and passively 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins But first: a reminder We often characterize a carbon atom by specifying how many hydrogens are attached to it –CH3 is methyl –CH2– is methylene –CH– is methine | 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins Quick iClicker quiz… 1. The standard form of the free-energy equation is (a) H = G - TS (b) G = H - TS (c) PV = nRT (d) none of the above. 08/29/2013 Amino Acids and Proteins

iClicker quiz question 2 2. A reaction in which reactants are spontaneously being converted to products is (a) unlikely (b) endergonic (c) exergonic (d) endothermal (e) exothermal 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins Special case: proline Proline isn’t an amino acid: it’s an imino acid Hindered rotation around bond between amine N and alpha carbon is important to its properties Tends to abolish -helicity because of that hindered rotation 08/29/2013 Amino Acids and Proteins

The simplest amino acids Glycine Alanine These are moderately nonpolar methyl 08/29/2013 Amino Acids and Proteins

Branched-chain aliphatic aas Valine Isoleucine Seriously nonpolar isopropyl Leucine 08/29/2013 Amino Acids and Proteins

Hydroxylated, polar amino acids Serine Threonine hydroxyl 08/29/2013 Amino Acids and Proteins

Amino acids with carboxylate side chains Aspartate Glutamate carboxylate methylene 08/29/2013 Amino Acids and Proteins

Amino Acids with amide side chains asparagine glutamine amide Note: these are uncharged! Don’t fall into the trap! 08/29/2013 Amino Acids and Proteins

Sulfur-containing amino acids Cysteine Methionine sulfhydryl Two differences: (1) extra methylene (2) methylated S 08/29/2013 Amino Acids and Proteins

Positively charged side chains Lysine Guani- dinium Arginine 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins Aromatic Amino Acids Phenylalanine Tyrosine phenyl 08/29/2013 Amino Acids and Proteins

Histidine: a special case imidazole 08/29/2013 Amino Acids and Proteins

Tryptophan: the biggest of all indole 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins Chirality Remember: any carbon with four non-identical substituents will be chiral Every amino acid except glycine is chiral at its alpha carbon Two amino acids (ile and thr) have a second chiral carbon: C 08/29/2013 Amino Acids and Proteins

Ribosomally encoded amino acids are L-amino acids All have the same handedness at the alpha carbon The opposite handedness gives you a D-amino acid There are D-amino acids in many organisms Bacteria incorporate them into structures of their cell walls Makes those structures resistant to standard proteolytic enzymes, which only attack amino acids with L specificity 08/29/2013 Amino Acids and Proteins

The CORN mnemonic for L-amino acids Imagine you’re looking from the alpha hydrogen to the alpha carbon The substituents are, clockwise: C=O, R, N: 08/29/2013 Amino Acids and Proteins

Abbreviations for the amino acids 3-letter and one-letter codes exist All the 3-letter codes are logical Most of the 1-letter codes are too 6 unused letters, obviously U used for selenocysteine O used for pyrrolysine B,J,Z are used for ambiguous cases: B is asp/asn, J is ile/leu, Z is glu/gln X for “totally unknown” http://www.chem.qmul.ac.uk/iupac/AminoAcid/A2021.html 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins Acid-base properties -amino acids take part in a variety of chemical reactivities, but the one we’ll start with is acid-base reactivity The main-chain carboxylate and amine groups can undergo changes in protonation Some side chains can as well 08/29/2013 Amino Acids and Proteins

Letters A-F: acid-base properties Amino Acid Side-chain 3-lett abbr. 1-let pKa, COO- pKa, NH3+ alanine CH3 ala A 2.4 9.9 * asx B cysteine CH2SH cys C 1.9 10.7 aspartate CH2COO- asp D 2.0 glutamate (CH2)2COO- glu E 2.1 9.5 phenyl-alanine CH2-phe phe F 2.2 9.3 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins Letters G-L Amino Acid Side-chain 3-lett abbr. 1-let pKa, COO- pKa, NH3+ glycine H gly G 2.4 9.8 histidine -CH2-imidazole his 1.8 9.3 isoleucine CH(Me)Et ile I 2.3 Ile/leu * lex? J 9.7-9.8 lysine (CH2)4NH3+ lys K 2.2 9.1 leucine CH2CHMe2 leu L 9.7 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins Letters M-S pyrrolysine methionine (CH2)2-S-Me met M 2.1 9.3 asparagine CH2-CONH2 asn N 8.7 pyrrol- lysine see above pyl O 2.2 9.1 proline (CH2)3CH (cyc) pro P 2.0 10.6 glutamine (CH2)2CONH2 gln Q arginine (CH2)3-guanidinium arg R 1.8 9.0 serine CH2OH ser S 9.2 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins Letters T-Z threonine CH(Me)OH thr T 2.1 9.1 seleno- cysteine CH2SeH sec U 1.9 10.7 valine CH(Me)2 val V 2.3 9.7 tryptophan CH2-indole trp W 2.5 9.4 unknown Xaa X tyrosine CH2-Phe-OH tyr Y 2.2 9.2 glu/gln (CH2)2-COX glx Z 08/29/2013 Amino Acids and Proteins

Remembering the abbreviations A, C, G, H, I, L, M, P, S, T, V easy F: phenylalanine sounds like an F R: talk like a pirate D,E similar and they’re adjacent N: contains a nitrogen W: say tryptophan with a lisp Y: second letter is a Y Q: almost follows N, and gln is like asn You’re on your own for K,O,J,B,Z,U,X 08/29/2013 Amino Acids and Proteins

Do you need to memorize these structures? Yes, for the 20 major ones (not B, J, O, U, X, Z) The only other complex structures I’ll ask you to memorize are: DNA, RNA bases Ribose, glucose, glyceraldehyde Cholesterol, stearate, palmitate A few others I won’t enumerate right now. 08/29/2013 Amino Acids and Proteins

How hard is it to memorize the structures? Very easy: G, A, S, C, V Relatively easy: F, Y, D, E, N, Q Harder: I, K, L, M, P, T Hardest: H, R, W Again, I’m not asking you to memorize the one-letter codes, but they do make life a lot easier. 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins iClicker question #3 3. What amino acids are in ELVIS? (a) asp - lys - val - ile - ser (b) asn - lys - val - ile - ser (c) glu - leu - val - ile - ser (d) glu - lys - val - ile - ser (e) Thank you very much. (25 seconds) 08/29/2013 Amino Acids and Proteins

Amino Acids and Proteins … and another 4. How many of the twenty plentiful, ribosomally encoded amino acids have exactly one chiral center? (a) zero (b) one (c) seventeen (d) eighteen (e) twenty 08/29/2013 Amino Acids and Proteins

Main-chain acid-base chemistry Deprotonating the amine group: H3N+-CHR-COO- + OH-  H2N-CHR-COO- + H2O Protonating the carboxylate: H3N+-CHR-COO- + H+  H3N+-CHR-COOH Equilibrium far to the left at neutral pH First equation has Ka=1 around pH 9 Second equation has Ka=1 around pH 2 08/29/2013 Amino Acids and Proteins

Why does pKa depend on the side chain? Opportunities for hydrogen bonding or other ionic interactions stabilize some charges more than others More variability in the amino terminus, i.e. the pKa of the carboxylate group doesn’t depend as much on R as the pKa of the amine group 08/29/2013 Amino Acids and Proteins

When do these pKa values apply? The values given in the table are for the free amino acids The main-chain pKa values aren’t relevant for internal amino acids in proteins The side-chain pKa values vary a lot depending on molecular environment: a 9.4 here doesn’t mean a 9.4 in a protein! 08/29/2013 Amino Acids and Proteins

How do we relate pKa to percentage ionization? Derivable from Henderson-Hasselbalch equation If pH = pKa, half-ionized One unit below: 91% at more positive charge state, 9% at less + charge state One unit above: 9% / 91% 08/29/2013 Amino Acids and Proteins

Don’t fall into the trap! Ionization of leucine: pH 1.3 2.3 3.3 8.7 9.7 10.7 %+ve 91 50 9 % neutral %-ve Main species NH3+-CHR-COOH NH3+CHR-COO- NH3+ CHR-COO- NH2-CHR-COO- 08/29/2013 Amino Acids and Proteins

Side-chain reactivity Not all the chemical reactivity of amino acids involves the main-chain amino and carboxyl groups Side chains can participate in reactions: Acid-base reactions Other reactions In proteins and peptides, the side-chain reactivity is more important because the main chain is locked up! 08/29/2013 Amino Acids and Proteins