Amino Acids and Peptides II Andy Howard Introductory Biochemistry Fall 2009, IIT

09/03/09 Biochemistry: Amino Acidsp. 2 of 47 Acids, bases, amino acids We’ll look at the acid-base properties of amino acid main chains and side chains We’ll examine other properties of amino acids, and begin to see how oligomers and polymers of amino acids work

09/03/09 Biochemistry: Amino Acidsp. 3 of 47 Plans Chirality Abbreviations Acid/base chemistry Side-chain reactivity Peptides and proteins Side-chain reactivity in context Disulfides

09/03/09 Biochemistry: Amino Acidsp. 4 of 47 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 

09/03/09 Biochemistry: Amino Acidsp. 5 of 47 All have the same handedness at the alpha carbon The opposite handedness gives you a D-amino acid Ribosomally encoded amino acids are L-amino acids

09/03/09 Biochemistry: Amino Acidsp. 6 of 47 Do D-amino acids ever occur in organisms? Yes: 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

09/03/09 Biochemistry: Amino Acidsp. 7 of 47 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:

09/03/09 Biochemistry: Amino Acidsp. 8 of 47 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 pyrrollysine –B,J,Z are used for ambiguous cases: B is asp/asn, J is ile/leu, Z is glu/gln –X for “totally unknown”

09/03/09 Biochemistry: Amino Acidsp. 9 of 47 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

09/03/09 Biochemistry: Amino Acidsp. 10 of 47 Letters A-F: acid-base properties Amino Acid Side- chain 3-lett abbr. 1- let pK a, COO - pK a, NH 3 + alanine CH 3 alaA *asxB cysteineCH 2 SH cysC aspartateCH 2 COO - aspD glutamate (CH 2 ) 2 COO - gluE phenyl- alanine CH 2 -phepheF2.29.3

09/03/09 Biochemistry: Amino Acidsp. 11 of 47 Letters G-L Amino Acid Side- chain 3-lett abbr. 1- let pK a, COO - pK a, NH 3 + glycine HglyG histidine -CH 2 - imidazole hisH isoleucineCH(Me)Et ileI Ile/leu * lex?J lysine (CH 2 ) 4 NH 3 + lysK l eucine CH 2 CHMe 2 leuL2.39.7

09/03/09 Biochemistry: Amino Acidsp. 12 of 47 Letters M-S methionine (CH 2 ) 2 -S-Me metM asparagine CH 2 -CONH 2 asnN pyrrol- lysine see above pylO proline (CH 2 ) 3 CH (cyc) proP glutamine (CH 2 ) 2 CONH 2 glnQ arginine (CH 2 ) 3 - guanidinium argR serineCH 2 OH serS2.29.2

09/03/09 Biochemistry: Amino Acidsp. 13 of 47 Letters T-Z threonine CH(Me)OH thrT seleno- cysteine CH 2 SeH SecU valineCH(Me) 2 valV tryptophanCH 2 -indole trpW unknown XaaX tyrosineCH 2 -Phe-OH tyrY glu/gln (CH 2 ) 2 -COX glxZ

09/03/09 Biochemistry: Amino Acidsp. 14 of 47 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

09/03/09 Biochemistry: Amino Acidsp. 15 of 47 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 –Cholesterol, stearate, palmitate –A few others I won’t enumerate right now.

09/03/09 Biochemistry: Amino Acidsp. 16 of 47 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.

09/03/09 Biochemistry: Amino Acidsp. 17 of 47 An iClicker question 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)

09/03/09 Biochemistry: Amino Acidsp. 18 of 47 … and another 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

09/03/09 Biochemistry: Amino Acidsp. 19 of 47 Main-chain acid-base chemistry Deprotonating the amine group: H 3 N + -CHR-COO - + OH -  H 2 N-CHR-COO - + H 2 O Protonating the carboxylate: H 3 N + -CHR-COO - + H +  H 3 N + -CHR-COOH Equilibrium far to the left at neutral pH First equation has K a =1 around pH 9 Second equation has K a =1 around pH 2

09/03/09 Biochemistry: Amino Acidsp. 20 of 47 Why does pK a 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 pK a of the carboxylate group doesn’t depend as much on R as the pK a of the amine group

09/03/09 Biochemistry: Amino Acidsp. 21 of 47 When do these pK a values apply? The values given in the table are for the free amino acids The main-chain pK a values aren’t relevant for internal amino acids in proteins The side-chain pK a values vary a lot depending on molecular environment: a 9.4 here doesn’t mean a 9.4 in a protein!

09/03/09 Biochemistry: Amino Acidsp. 22 of 47 How do we relate pK a to percentage ionization? Derivable from Henderson-Hasselbalch equation If pH = pK a, half-ionized One unit below: –90% at more positive charge state, –10% at less + charge state One unit above: 10% / 90%

09/03/09 Biochemistry: Amino Acidsp. 23 of 47 Don’t fall into the trap! Ionization of leucine: pH %+ve % neutral %-ve Main species NH 3 +- CHR- COOH NH 3 + CHR- COO - NH 2 - CHR- COO -

09/03/09 Biochemistry: Amino Acidsp. 24 of 47 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!

09/03/09 Biochemistry: Amino Acidsp. 25 of 47 Acid-base reactivity on side chains Asp, glu: side-chain COO - : –Asp sidechain pK a = 3.9 –Glu sidechain pK a = 4.1 –That means that at pH = 5.1, a glutamate will be ~90.9% charged Lys, arg: side-chain nitrogen: –Lys sidechain –NH 3 + pK a = 10.5 –Arg sidechain =NH 2 + pK a = 12.5

09/03/09 Biochemistry: Amino Acidsp. 26 of 47 Acid-base reactivity in histidine It’s easy to protonate and deprotonate the imidazole group

09/03/09 Biochemistry: Amino Acidsp. 27 of 47 Cysteine: a special case The sulfur is surprisingly ionizable Within proteins it often remains unionized even at higher pH

09/03/09 Biochemistry: Amino Acidsp. 28 of 47 Ionizing hydroxyls X–O–H  X–O - + H + Tyrosine is easy, ser and thr hard: –Tyr pK a = 10.5 –Ser, Thr pK a = ~13 Difference due to resonance stabilization of phenolate ion:

09/03/09 Biochemistry: Amino Acidsp. 29 of 47 Resonance-stabilized ion

09/03/09 Biochemistry: Amino Acidsp. 30 of 47 Other side-chain reactions Little activity in hydrophobic amino acids other than van der Waals Sulfurs (especially in cysteines) can be oxidized to sulfates, sulfites, … Nitrogens in his can covalently bond to various ligands Hydroxyls can form ethers, esters Salt bridges (e.g. lys - asp)

09/03/09 Biochemistry: Amino Acidsp. 31 of 47 Phosphorylation ATP donates terminal phosphate to side-chain hydroxyl of ser, thr, tyr ATP + Ser-OH  ADP + Ser-O-(P) Often involved in activating or inactivating enzymes Under careful control of enzymes called kinases and phosphatases

09/03/09 Biochemistry: Amino Acidsp. 32 of 47 Peptides and proteins Peptides are oligomers of amino acids Proteins are polymers Dividing line is a little vague: ~ aa. All are created, both formally and in practice, by stepwise polymerization Water eliminated at each step

09/03/09 Biochemistry: Amino Acidsp. 33 of 47 Growth of oligo- or polypeptide

09/03/09 Biochemistry: Amino Acidsp. 34 of 47 The peptide bond The amide bond between two successive amino acids is known as a peptide bond The C-N bond between the first amino acid’s carbonyl carbon and the second amino acid’s amine nitrogen has some double bond character

09/03/09 Biochemistry: Amino Acidsp. 35 of 47 Double-bond character of peptide

09/03/09 Biochemistry: Amino Acidsp. 36 of 47 The result: planarity! This partial double bond character means the nitrogen is sp 2 hybridized Six atoms must lie in a single plane: –First amino acid’s alpha carbon –Carbonyl carbon –Carbonyl oxygen –Second amino acid’s amide nitrogen –Amide hydrogen –Second amino acid’s alpha carbon

09/03/09 Biochemistry: Amino Acidsp. 37 of 47 Rotations and flexibility Planarity implies  = 180º, where  is the rotation angle about N-C bond Free rotations are possible about N-C  and C  -C bonds –Define  = rotation about N-C  –Define  = rotation about C  -C We can characterize main-chain conformations according to , 

09/03/09 Biochemistry: Amino Acidsp. 38 of 47 Ramachandran angles G.N. Ramachandran

09/03/09 Biochemistry: Amino Acidsp. 39 of 47 Preferred Values of  and  Steric hindrance makes some values unlikely Specific values are characteristic of particular types of secondary structure Most structures with forbidden values of  and  turn out to be errors

09/03/09 Biochemistry: Amino Acidsp. 40 of 47 How far from 180º can  vary? Remember what we said about the partial double bond character of the C-N main-chain bond That imposes planarity In practice it rarely varies by more than a few degrees from 180º.

09/03/09 Biochemistry: Amino Acidsp. 41 of 47 Ramachandran plot Cf. figures in text If you submit a structure to the PDB with Ramachandran angles far from the yellow regions, be prepared to justify them!

09/03/09 Biochemistry: Amino Acidsp. 42 of 47 How are oligo- and polypeptides synthesized? Formation of the peptide linkages occurs in the ribosome under careful enzymatic control Polymerization is endergonic and requires energy in the form of GTP (like ATP, only with guanosine): GTP + n-length-peptide + amino acid  GDP + P i + (n+1)-length peptide

09/03/09 Biochemistry: Amino Acidsp. 43 of 47 What happens at the ends? Usually there’s a free amino end and a free carboxyl end: H 3 N + -CHR-CO-(peptide) n -NH-COO - Cyclic peptides do occur Cyclization doesn’t happen at the ribosome: it involves a separate, enzymatic step.

09/03/09 Biochemistry: Amino Acidsp. 44 of 47 Reactivity in peptides & proteins Main-chain acid-base reactivity unavailable except on the ends Side-chain reactivity available but with slightly modified pK a s. Terminal main-chain pK a values modified too Environment of protein side chain is often hydrophobic, unlike free amino acid side chain

09/03/09 Biochemistry: Amino Acidsp. 45 of 47 Another iClicker question What’s the net charge on ELVIS at pH 7? (a) 0 (b) +1 (c) -1 (d) +2 (e) -2

09/03/09 Biochemistry: Amino Acidsp. 46 of 47 Disulfides In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains

09/03/09 Biochemistry: Amino Acidsp. 47 of 47 What could this do? Can bring portions of a protein that are distant in amino acid sequence into close proximity with one another This can influence protein stability