Lehninger Principles of BiochemistryFourth Edition

Amino Acids, Peptides, and ProteinsCopyright 2004 by W. H. Freeman & CompanyDavid L. Nelson and Michael M. Cox

Chemical Foundations

Stereochemistry Configuration & Conformation

Stereochemistry: the arrangement of the molecules atoms in 3-D space.

Configuration: Fixed structural arrangement of atoms in a molecule.

Conformation: Spatial arrangements of atoms in a molecule that can come about through free rotation of the atoms about a single bond (without breaking any bonds).

R vs S D vs L cis (Z) vs trans (E)

cis/trans isomers are different compounds with different properties. R/L isomers share similar chemical, but different biological properties.

Interactions between biomolecules are stereospecific.

Stereoisomers: Molecules with the same chemical bonds (chemical formula), but different stereochemistry (configuration).

Vitamin A (trans-Retinol) cis-retinol10-12 secConformational change of rhodopsin (photoreceptor)http://www.chemistry.wustl.edu/~edudev/LabTutorials/Vision/Vision.htmlElectrical impulsesBrain (I see the light!)

Chirality

A molecule with n chiral centers 2n stereoisomers.

Conformers Not isomers. In different conformational states: (1) eclipsed vs staggered (gauche, anti),

(2) chair form vs boat form

Protein conformers:

A protein (trypsin, etc.) can exist in different conformations resulting from conformational change induced by binding of a ligand (activator, inhibitor) etc.

regulation of enzyme activity

Interactions between biomolecules are stereospecific.

[1] Amino Acids

(1) Description The standard amino acids are -amino acids. primary amino group (NH2) carboxylic acid group (COOH)

Proline is an exception with a secondary amino group, but, it is still referred to as an -amino acid.

Amino acids also exist in a zwitterionic form at pH 7. The amino group is protonated (pKa ~9.4). The carboylic acid group is deprotonated (carboxylate; pKa ~2.2).

Amino acid structures differ at the side chain (R-groups). Abbreviations: three or one letter codes Amino acids (except glycine) have chiral centers: Rotate the plane of plane-polarized light and are optically active.

Amino acid carbons are named in sequenceusing the Greek alphabet (, , , , ) starting at the carbon between the carboxyl and amino groups.

Configuration of biological chiral compounds is defined in relation to glyceraldehyde (L- & D-) D - dextrorotatory (rotating light to the right) L - levorotatory (rotating light to the left)

BUT L or D designation for an amino acid does NOT reflect its ability to rotate plane polarized light in a particular direction!

The amino acids in proteins are L isomers.

(2) Amino Acids: Structural Classification (Table 3-1, p. 78)

Cystine residues provide structural stability of proteins through intramolecular or intermolecular disulfide bonds. OxidationReduction

Disulfide bonds and permanent waving.

Box 4-2, p.127

Non-standard (Non-canonical) amino acids: These are produced by post-translational modification, and not genetically coded, except selenocystein (Sec), which is now accepted as the 21st amino acids.

D-amino acids are found in a few small peptides, including some peptides of bacterial cell walls and certain antibiotics (such as penicillin).

D-GluD-Ala

Alexander Fleming discovers penicillin: http://www.pbs.org/wgbh/aso/databank/entries/dm28pe.html

Penicillin binds at the active site of the transpeptidase enzyme that cleaves D-Ala-D-Ala and thus cross-links the peptidoglycan strands of the bacterial cell wall. Penicillin binds the enzyme by mimicking the D-Ala-D-Ala residues that would normally bind to this site. Therefore, the bacterial cell wall loses integrity and is susceptible to host defences, or simply bursts.

(3) Amino Acid Properties: pKa, pI and TitrationsIn water, amino acid exists as zwitterions (hybrid ion). Amino acids as acids and bases. (ampholytes)

(a) Acidity and Basicity of amino acids

pKa for the COOH group in amino acids is 2 ~ 2.3, two pH units lower than that of ordinary aliphatic carboxylic acid (pKa of CH3COOH = 4.6). Glycine is 100 times more acidic than acetic acid. ppKa for the NH3+ group in amino acids is 9 ~ 10, one pH unit lower than that of ordinary aliphatic amine (pKa of CH3NH3+ = 10.6). TThe amino group of glycine is 10 times less basic than the amino groups of methylamine.

Perturbed pKa values of amino acid side chains.

Note that those pKa values for amino acid R groups in Table 3-1 are those measured as free form in solution.The pKa of any functional group is greatly affected by its chemical environment. There are ample examples where enzymes exploit the perturbed pKa of specific residues to carry out reactions.His57 (pKa = 12) in chymotrypsin (p.218, Lehninger): H-bond with Asp102His303 (pKa > 7.8) in chalcone synthase: ion pair with Cys164. HMG-CoA reductase E and D Glu-35 (pKa = 6.5) in lysozyme. Lys-NH3+ (pKa = 5.9) in acetoacetate decarboxylase Tyr and surrounding +ve residues in DNA gyrase .

COOH in a low polarity environment ionization is disfavoured. pKa COO - - +H3N ion pair ionization is stabilized via salt bridge. pKa of COOH , pKa of NH3+ . NH2 in a low polarity environment charged group is disfavoured pKa

(b) Titration Curve of Amino Acids

The pH at which a molecules net charge is zero is called the isoelectric point or the pI For two ionizable groups: pI = ? (such as carboxyl & amino) pKa1 + pKa2 pI = 2

Calculating pI Values for Amino Acids with a Charged R Group1. Use your knowledge of titration curves to figure out in which region the molecule will be neutral. (This involves drawing the titration curve and each associated species.)2. Find the pKa values that surround the neutral state.3. pI = (pKx + pKy)/2

pI is defined as the point at which there is no net charge on the molecule, or the molecule is neutral.

Lets take an example using a triprotic amino acid:Glutamate: pK1 = 2.18 (-COOH), pK2 = 8.95 (-NH3+) and in this case there will be a pKR = 4.25 (-COOH of R group)1. Draw the structure of Glu.(2. Draw the titration curve (pH vs. [OH-]) for Glu.)3. Where is the molecule neutral?4. How do we calculate the pI of Glu: (2.19+4.25)/2 = 3.22.

A good buffer at ~ pH 6. pI =

Histidine

A midterm questionThe amino acid aspartic acid has three ionizable groups, with pKa values of 1.9, 3.7, and 9.6. What is the average net charge of aspartic acid at pH 4.3 ?

(4) Functions (Lehninger 428-429)1. Chemical Messengers: Neurotransmitters are roughly divided into small molecules & peptidic (neuropetides), these are further classified as inhibitor and excitatory.Inhibitory Neurotransmitters Glycine (-OOC-CH2-NH3+) binds a receptor that depolarizes the synapse by Cl- release involved in motor and sensory functions -Aminobutyric acid (-OOC-(CH2)3-NH3+) GABA Glutamic acid derivative Most common neurotransmitter in brain Huntingtons disease - altered levels (B) Excitatory Neurotransmitters Glutamic acid and Aspartic acid: learning and memory Epinephrine (=adrenaline) and norepinephrine are derived from tyrosine (Fig 22-29, p. 860)

2. Monomeric subunits of proteins All proteins are composed of some or all of the 20 "standard" amino acids two new amino acids have been recently discovered!Discovery of 21st amino acid: (Selenocystein)Discovery of 22nd amino acid: (Pyrrollysine) http://unisci.com/stories/20022/0524025.htm

Energy metabolites many are essential nutrients and can be used as precursors to other molecules.

[2] Peptides and Proteins (MW > 10,000): Polymers of amino acids Energetically unfavourable (G > 0) Couple with energetically favourable reaction(s) (Leaving group activation) (Lehninger Fig 27-14, p.1052.) Peptide synthesis:

Serylglycyltyrosylalanylleucine.Ser-Gly-Tyr-Ala-LeuSGTAL N-terminusC-terminus

Name: Full name, Three letter codes One letter codes

pI value

Peptide bonds are stable: t1/2 = 7 yrs in cells due to high G of the hydrolysis reaction.

Peptides (and proteins) have their unique pI values depending on the a.a. compositions.

Biologically active peptides: oxytocin, bradykinin, vasopressin, etc. insulin, glucagon,

Monogamy and vasopressin and oxytocinhttp://serendip.brynmawr.edu/bb/neuro/neuro02/web1/afarrell.htmlVasopressin Oxytocin

Rough estimation of MW of proteins: Number of a. a. residues 110

Conjugated proteins: Contain permanently-attached (via covalent or coordination bonds), non-amino acid, chemical groups called prosthetic groups.

Multisubunit proteins. Subunits: noncovalent association, and subunits of hemoglobin Chains: covalently bonded (via disulfide bonds), A and B chains of insulin Hierarchy of protein structures:

[3] The Covalent Structure of Proteins Sequence (Primary structure, covalent linkages of amino acids) determines Tertiary structure (3-D structure) Function & Cellular location (enzymes, transport, movement, antibodies, receptors, structural roles) (1) Protein sequencing (a) N-terminal determination with FDNB (1-fluoro-2,4-dinitrobenzene) or Dansyl chloride (b) Edman degradation

(2) Sequencing of a large protein by overlapping method Amino acid composition Reduction of disulfide bonds & acetylation of Cys Fragmentation : enzymatic or chemical (Table 3-7) Purification & sequencing of the peptides Protein sequence by overlapping peptide sequences.

Reduction of disulfide bonds & acetylation of Cys

Enzymatic Fragmentation

(3) Deduced amino acid sequences from DNA sequences

Go to: http://www.ncbi.nlm.nih.gov/entrez/ Select for

Get 17 hits Click on the gene accession number

6: DQ275627 (gene accession number)Physcomitrella patens chalcone synthase mRNA, complete cds gi|94553859|gb|DQ275627.2|[94553859]Online search for nucleotide and protein sequences Nucleotide

Physcomitrella chalcone synthase

LOCUS DQ275627 1656 bp mRNA linear PLN 04-MAY-2006DEFINITION Physcomitrella patens chalcone synthase mRNA, complete cds.ACCESSION DQ275627VERSION DQ275627.2 GI:94553859

Protein function 3-D structure Sequence

The goal is to learn, from sequence, as much information as possible on its structure, function, and its evolutionary history.

[4] Protein Sequences and EvolutionGenes and proteins from closely related organisms should be similar.

The sequences increasingly diverge as the evolutionary distance between two organisms increases.

Conserved a.a. residues: amino acid residues essential for function and structure are conserved throughout the evolution.

Variable residues: Those less important vary over time. polymorphism

Protein family: A group of proteins with significant sequence similarity (>25%), and/or with similar structure and function. Likely share common evolutionary origin. Ser proteinases: chymotrypsin, trypsin, elastase, etc. Cytochrome P450 family.PROSITE: http://www.expasy.org/prosite/InterPro: http://www.ebi.ac.uk/interpro/ Homologs, paralogs (gene duplication), orthologs (speciation)

Multiple sequence alignment & Conservative Substitution

Homologs are identified by comparing multiple sequences of a protein from different organisms.

Multiple sequences are aligned to maximize the sequence similarity.

Conservative substitution by a chemically similar a.a. residue (Asp and Glu, Lys and Arg, Trp and Phe, etc) is given a high score during alignment according to the scoring system such as Blosum62.

Scores are based on how frequently an amino acid was replaced by each of the other amino acids.

Signature Sequence: helps distinguish/separate one group of organism from others.Example 1) A signature seq in the EF-1/EF-Tu protein family.

Example 2) A signature sequence (GFGPG) in the plant chalcone synthase (CHS) family. CHS 362 --------- GEGLEWGVLF GFGPGLTIET STS 362 --------- GEGLDWGVLF GFGPGLTIET KAS II 376 DLDFVPHEA -SGMEYTLCN SFGFGGTNGS PKS KAS 427 DWAGLDIRL RPHPRRAAVS GFGYGGTVAHKAS III 380 --------- VKPGNTTATS GFGAGLTWGS

Evolutionary tree derived from amino acid sequence comparison