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Mecanismos de Bioquímica · Biomolecules

Aminoácidos, peptídeos e proteínas

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O que você vai aprender

  • Assign the charge state of an amino acid or peptide at a given pH from its pKa values, and calculate isoelectric points and fractions ionised.
  • Draw the arrow-pushing mechanisms that make peptide bonds from activated carboxyl groups and break them in hot aqueous acid.
  • Explain disulfide formation and cleavage as ionic thiol–disulfide exchange, and relate covalent and noncovalent interactions to the levels of protein structure.
  • Use the chemistry of Sanger, Edman and cyanogen bromide reagents, and of trypsin specificity, to read the sequence of a peptide.

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An amino acid is a set of acid–base groups

Every amino acid carries an α-carboxyl group (pKa about 2) and an α-ammonium group (pKa about 9–10). Between those two values the dominant form is the zwitterion, ⁺H₃N–CHR–COO⁻, with net charge zero; the un-ionised form H₂N–CHR–COOH is never significant in water. Seven side chains also ionise. Standard textbook values: Asp 3.65, Glu 4.25, His 6.00, Cys 8.18, Tyr 10.07, Lys 10.53, Arg 12.48. A group is mostly protonated when the pH is below its pKa and mostly deprotonated when the pH is above it, and the Henderson–Hasselbalch equation, pH = pKa + log([base]/[acid]), gives the exact ratio. The α-carboxyl group (pKa 2.34 in glycine) is far more acidic than acetic acid (4.76) because the neighbouring ammonium group pulls electron density away and stabilises the carboxylate by electrostatic attraction.

Finding the isoelectric point

The isoelectric point, pI, is the pH at which the average net charge is zero. Write the forms in order of deprotonation, find the one with net charge zero, and average the two pKa values on either side of it. Glycine: (2.34 + 9.60)/2 = 5.97. Aspartic acid: the neutral form lies between the α-carboxyl and the side-chain carboxyl, so pI = (1.88 + 3.65)/2 = 2.77. Lysine: the neutral form lies between the α-ammonium and the side-chain ammonium, so pI = (8.95 + 10.53)/2 = 9.74. Histidine: pI = (6.00 + 9.17)/2 = 7.59. Averaging all three pKa values, or the α-carboxyl and α-ammonium of an acidic or basic amino acid, is the most common error. For a peptide, count every ionisable group: the N-terminal ammonium, the C-terminal carboxylate and each ionisable side chain; the internal α-groups are tied up in peptide bonds.

Making and breaking the peptide bond

The nitrogen lone pair of an amide is delocalised into the carbonyl group, so the C–N bond has partial double-bond character. The six atoms Cα–C(=O)–N(H)–Cα lie in one plane, rotation about C–N is slow, and almost every peptide bond is trans; bonds to proline, whose nitrogen is in a ring, have a much larger cis population. The same delocalisation makes amides the least reactive neutral carboxylic acid derivatives. Mixing an amine with a carboxylic acid only forms an ammonium carboxylate salt, so peptide synthesis first converts the carboxyl group into an activated ester (an N-hydroxysuccinimide ester) or an O-acylisourea (with DCC), whose good leaving group lets the amine add and the tetrahedral intermediate collapse. Breaking a peptide bond without an enzyme needs forcing conditions, normally 6 M HCl at about 110 °C for 24 h (hot strong base also works and is used to measure Trp): the carbonyl oxygen is protonated, water adds, the nitrogen is protonated and leaves as a neutral amine, which the acid at once converts into an ammonium ion. Those acid conditions convert Asn and Gln into Asp and Glu and destroy most of the Trp.

Disulfides and the levels of protein structure

A cysteine thiol (pKa 8.18) is a modest nucleophile, but its thiolate is excellent. Thiol–disulfide exchange is an SN2 reaction at sulfur: a thiolate attacks one sulfur of a disulfide and the other sulfur leaves as a thiolate. Because only the thiolate reacts, exchange speeds up as the pH rises toward the thiol pKa. Dithiothreitol (DTT) reduces disulfides completely because its second thiol closes a six-membered cyclic disulfide, an intramolecular step that strongly favours the products. Ellman's reagent (DTNB) uses the same exchange to release a yellow thiolate, TNB²⁻ (ε about 14,150 M⁻¹ cm⁻¹ at 412 nm), one per thiol. Disulfide formation is an oxidation: each sulfur goes from −2 in the thiol to −1 in the disulfide. Proteins are described at four levels: primary structure is the covalent sequence; secondary structure is the local backbone hydrogen-bonding pattern, such as the α-helix (C=O of residue i to N–H of residue i + 4) and the β-sheet; tertiary structure is the full three-dimensional fold of one chain, driven largely by burying hydrophobic side chains; quaternary structure is the assembly of several chains.

Reading a sequence with chemistry

Sanger's reagent, 1-fluoro-2,4-dinitrobenzene, tags free amino groups by nucleophilic aromatic substitution; after total acid hydrolysis the yellow DNP-amino acid identifies the N-terminal residue, and the side-chain amino group of every lysine reacts as well. Edman degradation is gentler and repetitive. Phenyl isothiocyanate adds to the N-terminal amine at about pH 9 to give a phenylthiocarbamoyl peptide; anhydrous trifluoroacetic acid then lets the thiourea sulfur attack the first peptide carbonyl, releasing an anilinothiazolinone (drawn here for glycine) and the intact peptide one residue shorter. The anilinothiazolinone is converted to the stable phenylthiohydantoin for identification. Each cycle is slightly less than 100 % efficient, so the fraction of chains still in step after n cycles is (efficiency)ⁿ, which limits a run to a few dozen residues. Longer chains are cut first: trypsin cleaves after Lys and Arg (not before Pro), and cyanogen bromide cleaves after Met, converting it into a C-terminal homoserine lactone. Overlapping fragments from two different cleavages then fix the order.

What to carry forward

Treat every amino acid and peptide as a collection of acid–base groups with known pKa values, and average the pKa values that flank the neutral form to get the pI. Peptide bonds form only from activated carboxyl groups and break only under forcing conditions such as hot 6 M HCl. Thiolates exchange with disulfides by SN2 at sulfur. Sequencing reagents are simply nucleophilic substitutions aimed at the N-terminal amine, at sulfur or at the first peptide carbonyl.

Problemas de prática (40)

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Desenhe o mecanismo 10

Peptide bonds: making and breaking

  • DCC coupling of N-acetylglycine with alanine methyl esterDraw how DCC activates the carboxyl group of N-acetylglycine as an O-acylisourea and how alanine methyl ester then forms the peptide bond, releasing N,N′-dicyclohexylurea.
  • Acid hydrolysis of glycylglycineDraw the acid-catalysed hydrolysis of the peptide bond in glycylglycine, using H₃O⁺ and water as the only acid and base, to give two glycine cations.
  • Forming a peptide bond from an N-hydroxysuccinimide esterDraw the addition of the alanine methyl ester amine to the activated NHS ester of N-acetylglycine, collapse of the tetrahedral intermediate with loss of the N-oxysuccinimide anion, and the final proton transfer that gives the neutral peptide bond.

Disulfides and protein structure

  • Dithiothreitol reduces cystineDraw the two thiol–disulfide exchanges by which dithiothreitol reduces cystine to two cysteines, ending with intramolecular ring closure to the cyclic disulfide of DTT.
  • Ellman's reagent detects a cysteine thiolDraw the thiol–disulfide exchange between cysteine thiolate and DTNB that releases the yellow TNB²⁻ anion, and the resonance that delocalises its charge into the para nitro group.
  • Glutathione thiolate exchanges with cystineDraw the SN2 attack of the glutathione thiolate on a sulfur of cystine that releases cysteine thiolate and forms the glutathione–cysteine mixed disulfide.

Sequencing chemistry

  • Edman coupling: phenyl isothiocyanate and Gly-AlaDraw the addition of the N-terminal amine of Gly-Ala to phenyl isothiocyanate and the proton transfers that give the phenylthiocarbamoyl peptide.
  • Edman cleavage: the thiourea sulfur cuts off the first residueDraw the anhydrous-acid cyclisation of phenylthiocarbamoyl-Gly-Ala in which the thiourea sulfur attacks the first peptide carbonyl and releases alanine plus the anilinothiazolinone of glycine.
  • Sanger's reagent labels the N-terminus of Gly-AlaDraw the nucleophilic aromatic substitution in which the N-terminal amine of Gly-Ala displaces fluoride from 1-fluoro-2,4-dinitrobenzene through a Meisenheimer complex, giving the DNP-peptide.

Charge, pKa and pI

  • Titrating histidine with hydroxideDraw the two proton transfers that convert the +1 form of histidine into its −1 form, removing the imidazolium proton before the α-ammonium proton.

Preveja o produto 6

Peptide bonds: making and breaking

  • What survives total acid hydrolysis?Predict how side-chain amides and acid-sensitive residues change during total acid hydrolysis of a protein.

Charge, pKa and pI

  • Alanine in a pH 7 bufferPredict the dominant protonation state of a simple amino acid at physiological pH from its two pKa values.
  • Glutamate at physiological pHPredict the dominant form and net charge of an acidic amino acid at pH 7.4.

Disulfides and protein structure

  • Which residue hides in the core?Predict where a residue sits in a folded globular protein from the polarity of its side chain.

Sequencing chemistry

  • What does cyanogen bromide leave behind?Predict the fragment formed when cyanogen bromide cleaves a peptide at methionine.
  • Sanger analysis of Lys-GlyPredict the DNP-amino acid obtained when the N-terminal residue of a peptide is lysine.

Classificar 3

Peptide bonds: making and breaking

  • Which structure is Ala-Gly?Identify a dipeptide from its name using the N-terminus to C-terminus convention.
  • The resonance form behind peptide rigidityDistinguish the charge-separated resonance contributor of an amide from its tautomers.

Disulfides and protein structure

  • What level of structure is an α-helix?Classify a backbone hydrogen-bonding pattern as a level of protein structure.

Comparar 3

Peptide bonds: making and breaking

  • Which acyl donor makes a peptide bond fastest?Rank acyl derivatives by reactivity toward an amine using the basicity of their leaving groups.
  • Where are cis peptide bonds found?Compare cis/trans preferences of peptide bonds and identify the X–Pro bond as the one with a significant cis population.

Charge, pKa and pI

  • Rank amino acids by isoelectric pointRank acidic, neutral and basic amino acids by isoelectric point after computing each pI from its pKa values.

Calcular 12

Charge, pKa and pI

  • Isoelectric point of aspartic acidCalculate the isoelectric point of an acidic amino acid by identifying the two pKa values that flank its net-zero form.
  • Isoelectric point of glycineCalculate the isoelectric point of an amino acid without an ionisable side chain from its two pKa values.
  • How much histidine is protonated at pH 7?Use the Henderson–Hasselbalch equation to calculate the percentage of histidine side chains that are protonated at a given pH.
  • Isoelectric point of histidineCalculate the isoelectric point of histidine, whose side-chain pKa lies between the two α-group pKa values.
  • Isoelectric point of lysineCalculate the isoelectric point of a basic amino acid from the pKa values of its two ammonium groups.
  • Net charge of Asp-Lys-Glu-Ala at pH 7Calculate the net charge of a peptide at a given pH by counting only its termini and ionisable side chains.

Disulfides and protein structure

  • How much cysteine is thiolate at pH 7.4?Calculate the percentage of a cysteine thiol present as the reactive thiolate at physiological pH.
  • Oxidation state of sulfur in cystineAssign the oxidation state of sulfur in a disulfide and recognise disulfide formation as an oxidation.
  • Counting thiols with Ellman's reagentCalculate a thiol concentration from the absorbance of TNB²⁻ released in an Ellman assay.

Sequencing chemistry

  • How far can Edman degradation read?Calculate the cumulative yield of in-step chains after many Edman cycles from the per-cycle efficiency.
  • Count the tryptic fragmentsApply trypsin's cleavage rule, including the proline exception, to count the fragments of a peptide.

Peptide bonds: making and breaking

  • Molar mass of Gly-Ala-SerCalculate the molar mass of a peptide from free amino acid masses by subtracting one water per peptide bond.

Encontre o erro 1

  • Edman degradation gives nothingDiagnose why Edman degradation of an intact, pure peptide yields no PTH derivative in any cycle.

Interprete as evidências 1

  • Reading the glycine titration curveInterpret a point on the titration curve of glycine as the half-equivalence point of its carboxyl group.

Trabalhe de trás para frente 1

  • Assemble the sequence from two digestsWork back from overlapping trypsin and cyanogen bromide fragments to the full sequence of an octapeptide.

Planeje uma síntese 1

  • Keep two chains apart for sequencingPlan the reagent order that breaks a protein's disulfides and stops them from re-forming before sequencing.

Explique por quê 2

Charge, pKa and pI

  • Why is the α-carboxyl so acidic?Explain why the α-carboxyl group of an amino acid is more acidic than acetic acid.

Peptide bonds: making and breaking

  • Why is the peptide group flat?Explain the planarity and restricted rotation of the peptide bond in terms of amide resonance.

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