Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company CHAPTER 4 Amino Acids to accompany Biochemistry, 2/e by Reginald Garrett and Charles Grisham All rights reserved. Requests for permission to make copies of any part of the work should be mailed to: Permissions Department, Harcourt Brace & Company, 6277 Sea Harbor Drive, Orlando, Florida

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Outline 4.1: Amino Acids: Building Blocks of Proteins 4.2: Acid-Base Chemistry of Amino Acids 4.3: Reactions of Amino Acids 4.4: Optical Activity and Stereochemistry of Amino Acids 4.5: Spectroscopic Properties of Amino Acids 4.6: Separation and Analysis of a.a. Mixtures

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Amino Acids Building Blocks of Proteins

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Amino Acids Can Join Via Peptide Bonds

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 20 Common Amino Acids You should know names, structures, pKa values, 3-letter and 1-letter codes Non-polar amino acids Polar, uncharged amino acids Acidic amino acids Basic amino acids

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Uncommon Amino Acids We'll see some of these in later chapters Hydroxylysine, hydroxyproline - collagen Carboxyglutamate - blood-clotting proteins Pyroglutamate - bacteriorhodopsin Phosphorylated amino acids - signaling device

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 4.2 Acid-Base Chemistry Amino Acids are Weak Polyprotic Acids H 2 A + + H 2 O  HA 0 + H 3 O + K a1 = [ HA 0 ] [ H 3 O + ] __________________________ [H 2 A + ]

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company 4.2 Acid-Base Chemistry The second dissociation (the amino group in the case of glycine): HA 0 + H 2 O  A ¯ + H 3 O + K a2 = [ A ¯ ] [ H 3 O + ] _______________________ [ HA 0 ]

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company pK a Values of the Amino Acids You should know these numbers and know what they mean! Alpha carboxyl group - pK a = 2 Alpha amino group - pK a = 9 These numbers are approximate, but entirely suitable for our purposes.

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company pK a Values of the Amino Acids You should know these numbers and know what they mean Arginine, Arg, R: pK a (guanidino group) = 12.5 Aspartic Acid, Asp, D: pK a = 3.9 Cysteine, Cys, C: pK a = 8.3 Glutamic Acid, Glu, E: pK a = 4.3 Histidine, His, H: pK a = 6.0

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company pK a Values of the Amino Acids You should know these numbers and know what they mean Lysine, Lys, K: pK a = 10.5 Serine, Ser, S: pK a = 13 Threonine, Thr, T: pK a = 13 Tyrosine, Tyr, Y: pK a = 10.1

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Titration of Glycine

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Titration of Glutamic Acid

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company A Sample Calculation What is the pH of a glutamic acid solution if the alpha carboxyl is 1/4 dissociated? pH = 2 + log 10 [1] ¯¯¯¯¯¯¯ [3] pH = 2 + (-0.477) pH = 1.523

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Titration of Lysine

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Another Sample Calculation What is the pH of a lysine solution if the side chain amino group is 3/4 dissociated? pH = log 10 [3] ¯¯ ¯¯¯¯¯ [1] pH = (0.477) pH = = 11.0

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Reactions of Amino Acids Carboxyl groups form amides & esters Amino groups form Schiff bases and amides Side chains show unique reactivities –Cys residues can form disulfides and can be easily alkylated –Few reactions are specific to a single kind of side chain

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Stereochemistry of Amino Acids All but glycine are chiral L-amino acids predominate in nature D,L-nomenclature is based on D- and L- glyceraldehyde R,S-nomenclature system is superior, since amino acids like isoleucine and threonine (with two chiral centers) can be named unambiguously

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Spectroscopic Properties All amino acids absorb in infrared region Only Phe, Tyr, and Trp absorb UV Absorbance at 280 nm is a good diagnostic device for amino acids NMR spectra are characteristic of each residue in a protein, and high resolution NMR measurements can be used to elucidate three-dimensional structures of proteins

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company Separation of Amino Acids Mikhail Tswett, a Russian botanist, first separated colorful plant pigments by ‘chromatography’ Many chromatographic methods exist for separation of amino acid mixtures –Ion exchange chromatography –High-performance liquid chromatography

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company

Biochemistry 2/e - Garrett & Grisham Copyright © 1999 by Harcourt Brace & Company