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`
`NPS EX. 2028
`CFAD v. NPS
`IPR2015-01093
`
`

`
`Principles of Biochemistry
`
`Second Edition
`
`Albert L. Lehninger, David L. Nelson, and Michael M. Cox
`
`Copyright (TC) 1993, 1982 by Worth Publishers, Inc.
`
`All rights reserved
`
`Printed in the United States of America
`
`Library of Congress Catalog Card No. 91-67492
`ISBN: 0—87901-500-4
`
`Printing: 5 4 3 2
`
`Year: 97 96 95 94 93
`
`Development Editor: Valerie Neal
`
`Design: Malcolm Grear Designers
`Art Director: George Touloumes
`
`Project Editor: Elizabeth Geller
`Production Supervisor: Sarah Segal
`
`Layout: Patricia Lawson
`Picture Editor: Stuart Kenter
`
`Illustration Design: Susan Tilberry
`
`Illustrators: Susan Tilberry, Alan Landau, and Joan Waites
`Computer Art: Laura Pardi Duprey and York Graphic Services
`
`Composition: York Graphic Services
`Printing and binding: R.R. Donnelley and Sons
`Cover: The active site of the prnteolytic ensynie chynit-trypsin, show-
`ing the substrate (blue and pllrplel and the amino acid residues (red
`and orange) critical to catalysis. Determination of the rletailetl
`reaction inechsnism of this enzyme [dt-ascribed on pp. 223—'.’.2G}
`helped to establish the general principles of en '.?._YT!1e action.
`
`Froritispicce: A view of tobacco rihuiose-1,5~bisphuspl1ste carboxyluse
`(ruhisco 1. This en:r.yme is central to photosynthetic carbon dioxide
`fixation; it is the most abundant euzyine in the biosphere. Different
`subunits are shown in blues and gruys. Importimt active site residtles
`are shown in red. Sullhtes bound at the active site (an artifact of the
`
`crystallization procedure) are shown in yellow.
`
`Cover, frontispiece, and part opening images produced by Alisa Zapp (see
`Molecular Modeling credits, p. IC-4) and enhanced by Academy Arts.
`
`Illustration credits begin on p.
`copyright page.
`
`IC—1 and constitute a continuation of the
`
`Worth Publishers
`
`33 Irving Place
`
`New York, NY 10003
`
`Page 2
`
`Page 2
`
`

`
`CHAPTER
`
`-lino Acids and Peptides
`
`' are the most abundant macromolecules in living cells, occur-
`all cells and all parts of cells. Proteins also occur in great vari-
`'ugg'nd.=s of different kinds may be found in a single cell. More-
`glproteins exhibit great diversity in their biological function. Their
`_
`" role is made evident by the fact that proteins are the most
`is final products of the information pathways discussed in
`of this book. In a sense, they are the molecular instruments
`which genetic information is expressed. It is appropriate to
`he study of biological macromolecules with the proteins, whose
`erives from the Greek protos, meaning “first” or “foremost.”
`latively simple monomeric subunits provide the key to the struc-
`of the thousands of different proteins. All proteins, whether from
`
`5
`
`flare constructed from the same ubiquitous set of 20 amino acids,
`Hiéiitly linked in characteristic linear sequences. Because each of
`' mino acids has a distinctive side chain that determines its
`
`_
`
`"
`
`a1 properties, this group of 20 precursor molecules may be re-
`as the alphabet in which the language of protein structure is
`
`Proteins are chains of amino acids, each joined to its neighbor by a
`fie" type of covalent bond. What is most remarkable is that cells
`produce proteins that have strikingly different properties and ac-
`: .by joining the same 20 amino acids in many different combina-
`and sequences. From these building blocks different organisms
`kc such widely diverse products as enzymes, hormones, anti-
`5 the lens protein of the eye, feathers, spider webs, rhinoceros
`(_F1g- 5-1), milk proteins, antibiotics, mushroom poisons, and a
`, ‘‘..d Of Other substances having distinct biological activities.
`F1'°l3e1_I1 structure and function is the topic for the next four chap-
`I _th1s chapter we begin with a description of amino acids and the
`st bonds that link them together in peptides and proteins.‘
`
`Figure 5-1 The protein keratin is formed by all
`vertebrates. It is the chief structural component of
`hair, scales, horn, wool, nails, and feathers. The
`black rhinoceros is nearing extinction in the wild
`because of the myths prevalent in some parts of the
`world that a powder derived from its horn has aph-
`rodisiac properties. In reality, the chemical proper-
`ties are no different from those of powdered bovine
`hooves or human fingernails.
`
`Page 3
`
`

`
`
`
`112
`
`Part II Structure and Catalysis
`
`Amino Acids
`
`Proteins can be reduced to their constituent amino acids by a variet .;-
`methods, and the earliest studies of proteins naturally focused on .-'|
`free amino acids derived from them. The first amino acid to be disc"
`ered in proteins was asparagine, in 1806. The last of the 20 to be 1"on 3-]
`threonine, was not identified until 1938. All the amino acids have t 5|
`ial or common names, in some cases derived from the source F"
`which they were first isolated. Asparagine was first found in EI..'=;pa
`gus, as one might guess; glutamate was found in wheat gluten; ty
`sine was first isolated from cheese (thus its name is derived from .i
`Greek tyros, “cheese”); and glycine (Greek glykos, “sweet”) was I
`named because of its sweet taste.
`
`'
`
`Amino Acids Have Common Structural Features
`
`All of the 20 amino acids found in proteins have a carboxyl group .-'
`an amino group bonded to the same carbon atom (the oz carbon) {_
`it
`5-2). They differ from each other in their side chains, or R grou
`which vary in structure, size, and electric charge, and influence ;
`solubility of amino acids in water. When the R group contains a.
`tional carbons in a chain, they are designated ,8, 7, 5, 6, etc., proceed’
`out from the oz carbon. The 20 amino acids of proteins are often 1'oI"o
`to as the standard, primary, or normal amino acids, to distingu
`them from amino acids within proteins that are modified after ,
`proteins are synthesized, and from many other kinds of amino ucl
`present in living organisms but not in proteins. The standard am
`acids have been assigned three-letter abbreviations and one-let
`symbols (Table 5-1), which are used as shorthand to indicate the so I
`position and sequence of amino acids in proteins.
`1
`We note in Figure 5-2 that for all the standard amino acids exc
`one (glycine) the oz carbon is asymmetric, bonded to four different as
`-j
`stituent groups: a carboxyl group, an amino group, an R group, an;
`hydrogen atom. The oz-carbon atom is thus a3'chi1:a‘l:(;'ér'1'ter.(see *-
`:. ro-
`3—9). Because of the tetrahedral arrangement of the bonding or.
`around the oz-carbon atom of amino acids, the four different substi .
`ent groups can occupy two different arrangements in space, which if
`nonsuperimposable mirror images of each other (Fig. 5-3). These _j
`forms are called enantiomers or stereoisomers (see Fig. 3-9). "
`molecules with a chiral center are also optically active—i.e., Ll
`can rotate plane-polarized light, with the direction of the rotation I
`fering for different stereoisomers.
`
`Figure 5-3 (a) The two stereoisomers of alanine.
`L- and D-alanine are nonsuperimposable mirror
`images of each other. (b, c) Two different conven-
`tions for showing the configurations in space of
`stereoisomers. In perspective formulas (b) the
`wedge-shaped bonds project out of the plane of the
`paper, the dashed bonds behind it. In projection
`formulas (c) the horizontal bonds are assumed to
`project out of the plane of the paper, the Vertical
`bonds behind. However, projection formulas are
`often used casually without reference to stereo-
`chemical configuration.
`
`Page 4
`
`C00’
`
`*
`H3N— |3—H
`
`3 H
`
`Glycine
`
`C00’
`+
`3:
`H3N— —H
`
`Amino acid
`
`Figure 5-2 General structure of the amino acids
`found in proteins. With the exception of the nature
`of the R group, this structure is common to all the
`oz-amino acids. (Proline, because it is an imino acid,
`is an exceptional component of proteins.) The a car-
`bon is shown in blue. R (in red) represents the R
`group or side chain, which is different in each
`amino acid. In all amino acids except glycine
`(shown for comparison) the oz-carbon atom has four
`different substituent groups.
`
`
`
`L-Alanine
`
`
`
`Q00‘
`H313-—¢<H
`CH3
`L-Alanine
`
`(|300’
`H;.IiI—-U—I[
`:_L.H_...
`L-Alanine
`
`(a)
`
`(b)
`
`(c)
`
`Q00"
`H»¢«-KIH3
`CH3
`D-Alanine
`
`C1100"
`H—(l:—1?IH3
`CH3
`D-Alanine
`
`
`
`Page 4
`
`

`
`Chapter 5 Amino Acids and Peptides
`
`properties and conventions associated with the standard amino acids
`-
`‘I
`
`PK1
`(—COOH)
`
`PK2
`(—NH§ )
`
`PKR
`(R group)
`
`pl
`
`Hydropathy
`index*
`
`Occurrence
`in Proteins (%)T
`
`?H_l"J
`T-I—-( ‘ ---()H
`
`FC
`
`H._.OH
`D-G-1-yceraldehyde
`
`(‘.200
`:
`4
`
`[El-—? —-N'[-I3
`CH3
`D-Alanine
`
`‘('7-HU
`
`Ho-44¢ -H
`3&1-IZOH
`L-Glycersltlehyde
`
`(_:0(J
`r1_.,1§-—g".-H
`
`L-Alanine
`
`Figure 5-4 Steric relationship of the stereoisomers
`of alanine to the absolute configuration of L- and
`D-glyceraldehyde. In these perspective formulas, the
`carbons are lined up vertically, with the chiral atom
`in the center. The carbons in these molecules are
`numbered beginning with the aldehyde or carboxyl
`carbons on the end, or 1 to 3 from top to bottom
`as shown. When presented in this way, the R group
`of the amino acid (in this case the methyl group
`of alanine) is always below the a carbon. L-Amino
`acids are those with the a-amino group on the left,
`and D-amino acids have the a-amino group on the
`right.
`
`Page 5
`
`i_.‘Dm_bi'uing liydi-up!-1uIJici'ty sml hydmphilicity; can be used to predict. which amino
`b'e'f'ou'ml in an aqueous uJwi1'umnen1'. E— values] and which will be found in a
`'_iu environment H values). sea em; in-2. Ftjcrnl Kyte. .1. & Doolittle. er. uses}
`I61. 157, 1Dl'i—1.'J2.
`
`occurrence in over 200 proteins. From Klapper, MH. (1977) Biochem. Biophys. Res.
`78, 1018—1024.
`
`.
`
`’
`
`'_
`
`_
`«._=
`
`lB"'cl"a1ssificatiou and naming of stereoisomers is based on the ab-
`I-Ijilnfiguration of the four substituents of the asymmetric car-
`For this purpose a reference compound has been chosen, to
`-.013. er optically active compounds are compared. This refer-
`Pflund is the 3-carbon sugar glyceraldehy-de (Fig. 5-4}, the
`fiflgar to have an asymmetric carbon atom. The naming of
`4 al3.1.0l'l8 of both simple sugars and amino acids is based on the
`' Wfifigllration of glyc'e.ra1del'1yde, as established by x-ray dif-
`"3_B'«alysis. Th_e stereoisomers of all chiral compounds having at,
`atétion related to that of L-glyceraldehyde are designated 1. (fo
`ry, derlvecl from two, meaning ‘‘left”), and the stereoisomer
`__ ET-:;:"D‘g1yw1'aldehyde are designated a {for dextrorotatory, dei.
`-_ dextro, meaning “right”l. T-he symbols 1. and D thus refer to
`Solute configuration of the four substituents around the chiral
`
`Page 5
`
`

`
`114
`
`Part Il Structure and Catalysis
`
`
`
`Proteins Contain L-Amino Acids
`
`Nearly all biological compounds with a chiral center occur naturally in
`only one stereoisomeric form, either D or L. _’.l._‘he=-amino‘ a-cids*in1:irotei-1-.1.
`moleculesare the'L“ stereoisomers. D-Amino acids have been found unly
`in small peptides of bacterial cell walls and in some peptide autibiot.i¢'3
`(see Fig. 5-19).
`It is remarkable that the amino acids of proteins are all L ste1*eo130_
`mers. As we noted in Chapter 3, when chiral compounds are formed by.
`ordinary chemical reactions, a racemic mixture of D and 1. isomers 1-E.
`sults. Whereas the L and D forms of chiral molecules are difficult for a.
`chemist to distinguish and isolate, they are as different as nighl; and
`day to a living system. The ability of cells to specifically synthesize the.
`L isomer of amino acids reflects one of many extraordinary properties
`of enzymes (Chapter 8). The stereospecificity of the reactions catalyzed
`by some enzymes is made possible by the asymmetry of their active
`sites. The characteristic three-dimensional structures of proteins
`(Chapter 7), which dictate their diverse biological activities, require
`that all their constituent amino acids be of one stereochemical series_
`
`Amino Acids Are Ionized in Aqueous Solutions
`
`Amino acids in aqueous solution are ionized and can act as acids or
`bases. Knowledge of the acid—base properties of amino acids is ex-
`tremely important in understanding the physical and biological prop-
`erties of proteins. Moreover, the technology of separating, identifying,
`and quantifying the different amino acids, which are necessary steps
`in determining the amino acid composition and sequence of protein‘
`molecules, is based largely on their characteristic acid-base behavior.
`Those a-amino acids having a single amino group and a single
`carboxyl group crystallize from neutral aqueous solutionsias fully ion-
`
`ized species known as. .'fGerman for “hybrid ions”), each
`having both a positive and a negative charge (Fig. 5-5). These ions are
`electrically neutral and remain stationary in an electric field. The di-
`polar nature of amino acids was first suggested by the observation that
`crystalline amino acids have melting points much higher than those of
`other organic molecules of similar size. The crystal lattice of amino"
`acids is held together by strong electrostatic forces between. positively-
`and negatively charged functional groups of neighboring molecules,
`resembling the stable ionic crystal lattice of NaCl (see Fig. 4-6).
`
`Amino Acids Can Be Classified by R Group
`
`An understanding of the chemical properties of the standard amino
`acids is central to an understanding of much of biochemistry. The topic
`can be simplified by grouping the amino acids into classes based on the
`properties of their R groups (Table 5—1], in particular, their polarity
`or tendency to interact with water at biological pH {near pH 7.0J. The
`polarity of the R groups varies widely, from totally nonpolar or hydro-
`phobic (water-insouble) to highly polar or hydrophilic (water-soluble].
`The structures of the 20 standard amino acids are shown in FigI1T9
`5-6, and many of their properties are listed in Table 5-1. There 31'“
`five main classes of amino acids, those whose R groups are: nonpolar
`and aliphatic; aromatic {generally nonpolar); polar but uncharged?
`negatively charged; and positively charged. Within each class there
`are gradations of polarity, size, and shape of the R groups.
`
`Page 6
`
`“F00”
`H2N—C—H
`l:
`Nonionic
`form
`
`“[00
`.
`H3N—(|?—H
`R
`Zwitterionic
`form
`
`Figure 5-5 Nonionic and zwitterionic forms of
`amino acids. Note the separation of the + and ~
`charges in the zwitterion, which makes it an elec-
`tric dipole. The nonionic form does not occur in sig-
`nificant amounts in aqueous solutions. The zwitter-
`ion predominates at neutral pH.
`
`Page 6
`
`

`
`Chapter 5 Amino Acids and Peptides
`
`Nonpolar, aliphatic R groups
`
`C00’
`I
`
`+
`
`H3N—(|]—H
`H
`
`C00”
`I
`
`+
`
`H3N—Cl)‘—H
`CH3
`
`Alanine
`
`C00 '
`l
`
`+
`
`H3N—i—H
`cfia C11,,
`Valine
`
`H3KI—
`
`G00"
`I
`
`—H
`
`CH2
`
`Aromatic R groups
`COO’
`I
`éflg
`
`+
`
`COO _
`+
`I
`H3N—(1—H
`&H2
`L
`
`as
`
`' H
`
`Phenylalanine
`
`Tyrosine
`
`Tryptophan
`
`Positively charged R groups
`
`C00"
`I
`+
`H3N—C—H
`
`C00"
`I
`+
`H3N—Cl)—H
`CH2
`(IZHZ
`is
`$11 +
`(|3=NH2
`NH2
`
`C00-
`+ J;
`—-H
`H3N
`(Ia;
`J3-NH\
`(I43/“H
`H
`
`Arginine
`
`Histidine
`
`Negatively charged R groups
`C00‘
`C00-
`+
`+
`I
`I
`I-I3N—(iJ—H
`H3N——(l)—H
`(|3H2
`(EH2
`COO’
`
`' O0‘
`
`Aspartate
`
`Glutamate
`
`Figure 5-6 The 20 standard amino acids of pro-
`teins. They are shown with their amino and car-
`boxyl groups ionized, as they would occur at
`pH 7.0. The portions in black are those common to
`all the amino acids; the portions shaded in red are
`the R groups.
`
`COO"
`H3f{I—(E—H
`H_g|:_CH3
`CH2
`$3,
`Isoleucine
`
`H
`+ /(Ii _
`H,_l\|«I
`EH2
`I-I30-—— H2
`
`Leucine
`
`Polar, uncharged R groups
`
`coo‘
`H31iI—(|J—H
`CH2
`H
`
`IS
`
`Cysteine
`
`C|!OO'
`+
`H3N—‘C£3—H
`H— —0}{
`$113-
`Threonine
`
`(|300_
`+
`H3N——(|3—H
`
`CH20H
`
`Serine
`
`coo‘
`H3fiI—(l:—H
`(EH2
`(|3H2
`s
`5H3
`Methionine
`
`(|100'
`H35J—c|:—H
`(I-111,
`(EH2
`C
`/ %
`
`o
`Hm
`Glutamine
`
`-
`
`.
`
`'
`
`'
`
`Asparagine
`
`lfmri Aliphatic R Groups The hydrocarbon R groups in this
`- ammo acids are nonpolar and hydrophobic (Fig. 5-6). The
`e chains of alanine, valine, leucine, and isoleucine, with
`.-si31“.°I‘»1V§ shapes, are important in promoting hydrophobic in-
`Within protein structures. Glycine has the simplest amino
`iiure. Where it is present in a protein, the minimal steric
`9f the glycine side chain allows much more structural flexi-
`a etlfcte other amino acids. Proline represents the opposite
`- 0 X Pelme. The secondary amino (imino) group is held in a
`--
`..
`. Tmatlon that reduces the structural flexibility of the protein
`- Point.
`
`Page 7
`
`

`
`116
`
`Part 11 Structure and Catalysis
`
`6'3
`
`Tryptophan
`
`UV
`
`0::
`
`
`
`Tyrosine
`
`Absorbance
`
`
`Phenylalanine
`i
`{, I I
`23:: 240 250 260 270 280 290 360 310
`Wavelength (nm)
`
`_‘_
`([700 T
`H3N—(|3—H
`CH2—:_'"l
`Cysteine
`
`(FOOT
`H,,1¢1—(|:—H
`“H—CH2
`Cysteine
`
`H313
`
`coo’
`<|.'.oo
`(I: —H H3f\i—o—H
`ca.-—-a H CH2
`Cystine
`
`Figure 5-7 Comparison of the light absorbance
`spectra of the aromatic amino acids at pH 6.0. The
`amino acids are present in equimolar amounts
`(10’3 M) under identical conditions. The light ab-
`sorbance of tryptophan is as much as fourfold
`higher than that of tyrosine. Phenylalanine absorbs
`less light than either tryptophan or tyrosine. Note
`that the absorbance maximum for tryptophan and
`tyrosine occurs near a wavelength of 280 nm.
`
`Aromatic R Groups Phenylalanine, tyrosine, and tryptophan,
`with their aromatic side chains (Fig. 5-6), are relatively nonpolar (hy-
`drophobic). All can participate in hydrophobic interactions, which are
`particularly strong when the aromatic groups are stacked on one an-
`other. The hydroxyl group of tyrosine can form hydrogen bonds, and it
`acts as an important functional group in the activity of some enzymes.
`Tyrosine and tryptophan are significantly more polar than phenylala-
`nine because of the tyrosine hydroxyl group and the nitrogen of the
`tryptophan indole ring.
`Tryptophan and tyrosine, and to a lesser extent phenylalanine,
`absorb ultraviolet light (Fig. 5-7 and Box 5-1). This accounts for the
`characteristic strong absorbance of light by proteins at a wavelength of
`280 nm, and is a property exploited by researchers in the characteriza-
`tion of proteins.
`
`Polar, Uncharged R Groups The R groups of these amino acids (Fig.
`5-6) are more soluble in water, or hydrophilic, than those of the non-
`polar amino acids, because they contain functional groups that form
`hydrogen bonds with water. This class of amino acids includes serine,
`threonine, cysteine, methionine, asparagine, and glutamine.
`The polarity of serine and threonine is contributed by their hydroxyl
`groups; that of cysteine and methionine by their sulfur atom; and that
`of asparagine and glutamine by their amide groups.
`Asparagine and glutamine are the amides of two other amino acids
`also found in proteins, aspartate and glutamate, respectively, to which
`asparagine and glutamine are easily hydrolyzed by acid or base. Cys-
`teine has an R group (a thiol group) that is approximately as acidic as
`the hydroxyl group of tyrosine. Cysteine requires special mention for
`another reason. It is readily oxidized to form a covalently linked di-
`meric amino acid cal1ed~'e§7ns't'i1"1e,.in wliicH‘tvi{6__<_:§§11e_ine .molecules'are
`joined-by-a-disulfide-bridge. Disulfide bridges of this kind occur in
`many proteins, stabilizing their structures.
`
`Negatively Charged (Acidic) R Groups The two amino acids having
`R groups with a net negative charge at pH 7.0 are aspartate and
`glutamate, each with a second carboxyl group (Fig. 5-6). These amino
`acids are the parent compounds of asparagine and glutamine, respec-
`tively.
`
`Positively Charged (Basic) R Groups The amino acids in which the R
`groups have a net positive charge at pH 7.0 are lysine, which has a
`second amino group at the e position on its aliphatic chain; arginine,
`which has a positively charged guanidino group; and histidine, con-
`taining an imidazole group (Fig. 5-6). Histidine is the only standard
`amino acid having a side chain with a pKa near neutrality.
`
`Page 8
`
`
`
`Page 8
`
`

`
`Chapter 5 Amino Acids and Peptides
`
`117
`
`BOX 54
`
`Absorption of Light by Molecules: The Lambert—Beer Law
`
`Measurement of light absorption is an important
`tool for analysis of many biological molecules. The
`fraction of the incident light absorbed by a solution
`at a given wavelength is related to the thickness of
`the absorbing layer (path length) and the concen-
`tration of the absorbing species. These two rela-
`tionships are combined into the Lambert—Beer
`law, given in integrated form as
`I
`log 70 = ecl
`
`where 10 is the intensity of the incident light, I is
`the intensity of the transmitted light, 5 is the
`molar absorption coefficient (in units of liters per
`mole-centimeter), c the concentration of the ab-
`sorbing species (in moles per liter), and l the path
`length of the light-absorbing sample (in centime-
`ters). The Lambert—Beer law assumes that the in-
`cident light
`is parallel and monochromatic and
`that the solvent and solute molecules are randomly
`
`oriented. The expression log (I0/I) is called the ab-
`sorbance, designated A.
`It is important to note that each millimeter path
`length of absorbing solution in a 1.0 cm cell absorbs
`not a constant amount but a constant fraction of
`
`the incident light. However, with an absorbing
`layer of fixed path length, the absorbance A is di-
`rectly proportional to the concentration of the ab-
`sorbing solute.
`The molar absorption coefficient varies with the
`nature of the absorbing compound, the'solvent, the
`wavelength, and also with pH if the light-absorb-
`ing species is in equilibrium with another species
`having a different spectrum through gain or loss of
`protons.
`In practice, absorbance measurements are usu-
`ally made on a set of standard solutions of known
`concentration at a fixed wavelength. A sample of
`unknown concentration can then be compared with
`the resulting standard curve, as shown in Figure 1.
`
` I
`
`0
`
`0
`
`I
`
`I
`40
`
`i
`
`i
`80
`
`I_
`
`_L!_
`120
`
`I
`
`I
`160
`
`I
`
`1
`200
`
`Figure 1 Eight standard solutions containing
`known amounts of protein and one sample contain-
`ing an unknown amount of protein were reacted
`with the Bradford reagent. This reagent contains a
`dye that shifts its absorption maximum to 595 nm
`when it binds amino acid residues. The A595 (ab-
`sorbance at 595 nm) of the standard samples was
`plotted against the protein concentration to create
`the standard curve, shown here. The A595 of the
`unknown sample, 0.58, corresponds to a protein
`concentration of 122 pig/mL.
`
`Protein concentration (pig/mL)
`
`
`
`Cells Also Contain Nonstandard Amino Acids
`
`In addition to the 20 standard amino acids that are common in all
`Proteins, other amino acids have been found as components of only
`i‘-e1‘tain types of proteins (Fig. 5~8aJ. Each of these is derived from one
`Of: the 20 standard amino acids, in a modification reaction that occurs
`after the standard amino acid has been inserted into a protein. Among
`the nrmstandard amino acids are 4-hydrO'xypr0li-ne,- a derivative of
`Droline, and 5-hydroxylysine; the former is found in plant cel1—wall
`pmteins, and both are found in the fibrous protein collagen of con-
`nective tissues. N-Methyllysine is found in myosin, a contractile
`protein of muscle. Another important nonstandard amino acid is
`
`‘
`
`
`
`Page 9
`
`,
`
`
`
`Page 9
`
`

`
`
`
`118
`
`Part II Structure and Catalysis
`
`H3FI~—CH2—CH2—CH2—Cl3H—COO_ HZN C If CH2 CH2 CH2
`
`(IJH COO"
`
`Ornithine
`
`+NH3
`
`ll
`O H
`
`Citrulline
`
`(b)
`
`:y-cafl‘.i'OY§?‘"gl'at—amate, fourld in the blood-clotting protein prothrom-
`bin as well as in certain other proteins that bind Ca2+ in their biologi-
`cal function. More complicated is the nonstandard amino acid des-
`mosi_ne, a derivative of four separate lysine residues, found in the
`fibrous protein elastin. 'S_éleno(_:}7s‘t‘éi_‘ne contains selenium rather
`than the oxygen of serine, and is found in glutathione peroxidase and a
`few other proteins.
`Some 300 additional amino acids have been found in cells and have
`a variety of functions but are not substituents of proteins. Orni-thine
`and ‘citrulline (Fig. 5—8b) deserve special note because they are key
`intermediates in the biosynthesis of arginine and in the urea cycle.
`These pathways are described in Chapters 21 and 17, respectively.
`
`Amino Acids Can Act as Acids and as Bases
`
`When a crystalline amino acid, such as alanine, is dissolved in water, it
`exists in solution as the dipolar ion, or zwitterion, which can act either
`as an acid (proton donor):
`
`1?
`5‘
`R—C—COO‘ xi‘ R—-(|J—COO‘ + H*
`*NH3
`NH3
`
`or as a base (proton acceptor):
`
`‘F
`‘F
`R—(|3—COO‘ + H’“ ,1 R—C|J—COOH
`+NH3
`"NH3
`
`Substances having ‘this dual nature are'ampl1'o‘t’e’ri?:-and are often
`called ififimtesf from “amphoteric
`electrolytes.” A simple
`monoamino monocarboxylic a—amino acid, such as alanine, is actually
`a diprotic acid when it is fully protonated, that is, when both its car-
`boxyl group and amino group have accepted protons. In this form it has
`two groups that can ionize to yield protons, as indicated in the follow-
`ing equation:
`
`W 1%
`Ir
`R—(|‘.—C!OOI.I. ——”—» R—(|}—COO‘
`
`+NH3
`
`+NH3
`
`1%
`W
`=7
`» R—(|3—COO‘
`
`NH2
`
`Amino Acids Have Characteristic Titration Curves
`
`Titration involves the gradual addition or removal of protons. Figure
`5-9 shows the titration curve of the diprotic form of glycine. Each
`molecule of added base results in the net removal of one proton from
`
`Page 10
`
`in‘
`IN r—(|,‘. ($112
`11.0.‘ __ /_(1H—COO‘
`_N
`
`\H
`H’
`4—Hydroxyproline
`
`H31¢I—cH2—cH—cH2—CH2—cH—coo‘
`I H I
`+NH3
`5-Hydroxylysine
`
`CH3 NH CH2 CH2 CH9 CH«_, CH COO"
`I
`‘NI-1;,
`
`6-N-Methyllysine
`
`COO‘
`I
`-ooc—cH—cH._.—(|3H—-coo
`+NH_.,
`‘y-C arboxyglutamate
`
`..
`I-l3N
`
`C00‘
`
`NH3
`<c1«12;g—o§I
`coo"
`
`>CH—(Gm:l2-I5’
`I
`
`+N
`
`»/
`
`FUQC
`
`+
`
`C00 '
`H;,N
`Desmosine
`
`HSe—CH2—-C|}H—COO‘
`+NH3
`Selenocysteine
`
`(a)
`
`Figure 5-8 (a) Some nonstandard amino acids
`found in proteins; all are derived from standard
`amino acids. The extra functional groups are shown
`in red. Desmosine is formed from four residues of
`lysine, whose carbon backbones are shaded in gray.
`Selenocysteine is derived from serine. (b) Ornithine
`and citrulline are intermediates in the biosynthesis
`of arginine and in the urea cycle. Note that two
`systems are used to number carbons in the naming
`of these amino acids. The ca, [3, y system used for
`y-carboxyglutamate begins at the a carbon (see Fig.
`5-2) and extends into the R group. The oz-carboxyl
`group is not included. In contrast, the numbering
`system used to identify the modified carbon in
`4-hydroxyproline, 5-hydroxylysine, and 6-N-methyl-
`lysine includes the oz-carboxyl carbon, which is des-
`ignated carbon 1 (or C-1).
`
`Page 10
`
`

`
`
`
`mlec-Hie of arni.no acid. The plot has two distinct stages, each cor-
`O-ne finding to the removal oi" one proton firoin glycine. Each of the two
`tiesllzes ,.,._.~.(y.ini1l.e.~= in aliape the titration curve of a monoprotic acid,
`_"5.1"a%1ea_u,
`-.}L.-..:1-.ic acid {see Fig. 4-10), and can be analyzed iii the same
`:l__Jacy__ At very low pH,
`the predominant
`ionic species of glycine is
`.rH,.N———Cll-,;~-COOH, the fully protonated form. At the midpoint in
`‘ti-i,gJf‘i1'i5l. stage ofthe titration, in which the —COOH group of glycine
`110%35
`its
`proton,
`equimolar
`concentrations
`of
`proton-donor
`(_.,. H3N,C'[I5;—(1(JOIi} and proton-‘acceptor l_ " I-I,-.,N--_C.H2—CO_C) ' } Spg_
`cl-@531-epreHent.Ai'. the lnidpoint of a titration (see Fig. 4-11.), the pH is
`"equal to the pK,, of the protonated group being titrated. For glycine, the
`H at the midpoint is 2.34, thus its —COOH group has a 13K“ of 2.34.
`[Recall that pH and pK,, are simply convenient no1;a1.io'ns for proton
`concenti.'al.ion and the equilibrium Constant for ioliiization, respectively
`(Chapter 4). The pk], is a measure of the tendency of a group to give up
`a proton, with that tendency decreasing tenfold as the pK,, increases by
`one unit.J As the titration proceeds, another important point is reached
`at pH 5.97. Here there is a_1p6i3't{(.‘)'f!‘i'r;ff'1'é'c't'i'()'I1,‘iat which-removal-of'the
`first-proton is essentially complete, and removal of the second has just
`begun. At this pH the glycine is present largely as the dipolar ion
`+H3N-CH2—COO’. We shall return to the significance of this inflec—
`tion point in the titration curve shortly.
`The second stage of the titration corresponds to the removal of a
`proton from the —NH§“ group of glycine. The pH at the midpoint of this
`stage is 9.60, equal to the pK,, for the_—NH§“ group. The titration is
`complete at a pH of about 12, at which point the predominant form of
`glycine is H2N—CH2—COO’.
`From the titration curve of glycine we can derive several important
`pieces of information. First, it gives a quantitative measure of the pK,,
`of each of the two ionizing groups, 2.34 for the —COOH group and 9.60
`for the —NH§‘ group. Note that the carboxyl group of glycine is over
`100 times more acidic (more easily ionized) than the carboxyl group of
`acetic acid, which has a pK,, of 4.76. This effect is caused by the nearby
`positively charged amino group on the oz—carbon atom, as described in
`Figure 5-10.
`The second piece of information given by the titration curve of gly-
`cine (Fig. 5-9) is that this amino acid has two regions of buffering
`power (see Fig. 4-12). One of these is the relatively flat portion of the
`curve centered about the first pl?” of 2.34, indicating that glycine is a
`good buf'I"or near this pH. The other bui'l'e'ring zone extends for "-1.2 pH
`units centered around pH 9.60. Note also that glycine is not a good
`buffer at the pH of intracellular fluid or blood, about 7.4.-.
`The I--Iondcrson-Haaseibalch equation {Chapter 4) can be used to
`Ciilculatr-. the proportions of proton-donor and proton-acceptor species
`Oi Elyciiie required to make a buffer at a given pH within the buffering
`ranges ofglycine; it also makes it possible to solve other kinds of buffer-
`l31"0l1len'13 involving amino acids (see Box 4-2).
`
`Chapter 5 Amino Acids and Peptides
`
`Kin,
`CH2 %
`COOH
`
`NH3
`
`(|3oo‘
`
`13
`
`NH2
`
`(|300’
`
`:.-.-:
`
` .________....-_.._.__.________;.'
`
`
`0
`
`0:5
`
`1
`
`1.5
`
`2
`
`OH” (equivalents)
`
`Figure 5-9 The titration curve of 0.1 M glycine
`at 25 °C. The ionic species predominating at key
`points in the titration are shown above the graph.
`The shaded boxes, centered about pK1 = 2.34 and
`pK2 = 9.60, indicate the regions of greatest buffer-
`ing power.
`
`Figure 5-10 (a) Interactions between the a-amino
`and av-carboxyl groups in an ct-amino acid. The
`nearby positive charge of the —NH;;* group makes
`ionization of the carboxyl group more likely (i.e.,
`lowers the pK, for —COOH). This is due to a stabi-
`lizing interaction between opposite charges on the
`zwitterion and a repulsive interaction between the
`positive charges of the amino group and the depart-
`ing proton. (b) The normal pKn for a carboxyl
`group is approximately 4.76, as for acetic acid.
`
`Acetic acid
`
`Lu-Amino acid (glycine)
`+1TIH3
`-1TiI'I_-i
`H -L|.‘.—COOH LT‘ H—(|3—COO" + H F
`H
`
`H (
`
`pKn = 2.34
`
`CH,-COOH _‘-é CH,,—COO" + H+
`
`pKl : 476
`
`a)
`
`(b)
`
`Page 11
`
`
`
`Page 11
`
`

`
`
`
`120
`
`Part II Structure and Catalysis
`
`The Titration Curve Predicts the
`
`Electric Charge of Amino Acids
`
`Another important piece of information derived from the titration
`curve of an amino acid is the relationship between its net electric
`charge and the pH of the solution. At pH 5.97, the point of inflection
`between the two stages in its titration curve, glycine is present as its
`dipolar form, fully ionized but with no net electric charge (Fig. 5-9).
`This characteristic pH is called the isoelectric point.or isoelectric
`- pH; designated- pI- or pH1. For an amino acid such as glycine, which has
`no ionizable group in the side chain, the isoelectric point is the arith-
`metic-mean of the two pKa values:
`1
`P1 = §(PK1 + PK2)
`
`which in the case of glycine is
`
`pl = %(2.34 + 9.60) = 5.97
`
`As is evident in Figure 5-9, glycine has a net negative charge at any
`pH above its pl and will thus move toward the positive electrode (the
`anode) when placed in an electric field. At any pH below its pl, glycine
`has a net positive charge and will move toward the negative electrode,
`the cathode. The farther the pH of a glycine solution is from its isoelec-
`tric point, the greater the net electric charge of the population of gly-
`cine molecules. At pH 1.0, for example, glycine exists entirely as the
`form "'H;,N—CH3—COOH, with a net positive charge of 1.0. At pH
`2.34, where there is an equal mixture of ‘H3N—CH2—C0OH and
`“H3N—CH2—COO‘, the average or net positive charge is 0.5. The
`sign and the magnitude of the net charge of any amino acid at any pH
`can be predicted in the same way.
`This information has practical importance. For a solution contain-
`ing a mixture of amino acids, the different amino acids can be sepa-
`rated on the basis of the direction and relative rate of their migration
`when placed in an electric field at a known pH.
`1
`
`Amino Acids Differ in Their Acid—Base Properties
`
`The shared properties of many amino acids permit some simplifying
`generalizations about the acid-base behavior of different classes of
`amino acids.
`
`All amino acids with a single a-amino group, a single oz-carboxyl
`group, and an R group that does not ionize have titration curves resem-
`bling that of glycine (Fig. 5-9). This group of amino acids is character-
`ized by havi