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Modifications of Amino Acid Residues 1

Modifications of Amino Acid Residues 1

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Modifications of Amino Acid Residues: Part 1

Overview

AMINO ACID MODIFICATIONS

  • Disulfide cross-linking (between two cysteine residues).
  • Hydroxylation (the addition of a hydroxyl group).
  • Carboxylation (the addition of a carboxyl group).
  • Phosphorylation (the addition of a phosphate group).
  • Methylation (the addition of methyl groups)
  • Acetylation (the addition of acetyl groups)
  • Glycosylation (the addition of sugars such as glucosamine or mannose)
  • ADP-ribosylation (the addition of ADP-ribose)
  • Ubiquitination (the addition of one or more ubiquitins)

DISULFIDE BONDS

  • 2 Cys residues form disulfide linkages
  • Begin to produce 3-D shape of the protein
    (lower entropy of unfolded protein --- destabilize it)
  • Formation = oxidative rxn – H atoms on both Cys residues lost
  • "OIL RIG" (oxidation is loss of e- & reduction is gain of e-):
    cystine = cysteine minus "e"
  • Biochemical corollary: strong reducing reagents --- beta-mercaptoethanol & dithiotheitol (DTT) --- break disulfide linkages.

HYDROXYLATION

  • Addition of -OH to Pro or Lys residue (hydroxyproline/hydroxylysine)
  • Important part of collagen structure

CARBOXYLATION

  • Addition of -COOH group to glutamate residue (gamma-carboxyglutamate)
  • -COOH adds to gamma-carbon, which already has carboxylic acid
  • Glutamate carboxylation: integral to function clotting factors

PHOSPHORYLATION

  • Addition of phosphate group to Ser, Thr or Tyr residues
  • Phosphorylation of serine/threonine: most common AA modification
  • Size & (-) charge of phosphate group: causes slight change in protein shape
  • Reversible modification: conformational change = switch mechanism in protein activation

CLINICAL CORRELATION

Ascorbic acid (vitamin C)

  • Cofactor in hydroxylation
  • Vit. C deficiency: scurvy, pathologic collagen -->abnormal collagen fibers

Vitamin K

  • Cofactor in glutamate carboxylation
  • Vit. K deficiency: impairs prothrombin function (clotting protein) -->hemorrhage

Epinephrine (adrenaline)

  • Changes enzyme activity via serine & threonine phosphorylation
  • Many other hormones/neurotransmitters use phosphorylation to start signaling cascades

Full-Length Text

  • Here we will continue to learn about post-translational modifications of the peptide residues in a polypeptide and how they affect the structure and function of proteins.
    • This tutorial is one of two on modifications to amino acid residues, and here we will focus on some modifications of amino acid residues and their functional ability.
  • Draw a chain of 20 amino acids, which we represent with circles with lines connecting them; this chain represents the polypeptide's primary structure.
  • Label the N-terminus and C-terminus, which are the beginning and end terminals of the polypeptide chain.
    • We will define our polypeptide sequence as we go through the relevant amino acid modifications, so that we can use it to learn some of to co- and post-translational modifications to primary protein structure.

Before we draw these modifications, let's revisit the two important limitations of this particular tutorial:

  • We will only represent some of the many ways that amino acid residues can be modified in a protein, many more exist.
  • No single protein will have the number of modifications that we will show here; we show them this way here within a single polypeptide chain for simplicity.
  • Denote that we will focus on four modifications:
    • Disulfide cross-linking, which occurs between two cysteine residues.
    • Hydroxylation (the addition of a hydroxyl group).
    • Carboxylation (the addition of a carboxyl group).
    • Phosphorylation (the addition of a phosphate group).

Let's begin with disulfide bonds.

  • Denote that disulfide cross-links are where two cysteine residues form disulfide linkages.
    • Although they are part of primary protein structure, they begin to provide the framework for the 3-dimensional shape of the protein because they lower the entropy of the unfolded protein, and thus destabilize it.
  • Label amino acids 5 and 10 on our polypeptide chain as cysteine and draw a disulfide bond between them
    • Disulfide bonds form only between two cysteine residues because they have a sulfhydryl group as their side chain.
  • Indicate that creation of disulfide bridges is considered oxidative – the hydrogen atoms on both cysteine residues are lost.
  • Write that cysteine residues in a disulfide bond are called cystine.

Let's help ourselves remember this as follows:

  • First, recall that the acronym "OIL RIG" from general chemistry tells us that oxidation is loss of electrons and reduction is gain of electrons.
  • Next, recognize that cystine is cysteine without the middle "e", which helps clue us into the loss of electrons.
    • As a biochemical corollary, consider that strong reducing reagents such as beta-mercaptoethanol and dithiotheitol (DTT) break disulfide linkages.

Next we will look at hydroxylation.

  • Denote that hydroxylation is the addition of a hydroxyl group to a proline or lysine residue.
    • Proline and lysine may be hydroxylated to hydroxyproline and hydroxylysine.
  • Label amino acid 7 as proline and draw an OH group attached to it.
    • Hydroxylation of proline is an important part of collagen structure.
  • As a clinical correlation, denote that ascorbic acid (Vitamin C) is a cofactor in the hydroxylation of proline, which is why vitamin C deficiency results in scurvy in which pathologic collagen function occurs due to abnormal collagen fibers.
    • Interestingly, aside from its role as a cofactor for hydroxylating enzymes and other similar enzymatic reactions, vitamin C has no other known biological function (which brings into question its popularity as a cold remedy)!

Now let's look at carboxylation.

  • Denote that carboxylation is the addition of a carboxylic acid group to a glutamate residue to make gamma-carboxyglutamate.
    • The carboxyl group is added to the gamma-carbon of glutamate (the carbon with the carboxylic acid already attached), hence the name of the modified amino acid.
  • Label amino acid 14 as glutamate and draw a carboxylic acid group attached to it.
    • Glutamate carboxylation is integral to the function of some proteins, particularly clotting factors.
  • As a clinical correlate, write that in vitamin K deficiency, insufficient carboxylation impairs the function of pro-thrombin, an important clotting protein, which can result in hemorrhage.
    • This occurs because vitamin K is a cofactor for the carboxylation of glutamate.

Finally we will look at phosphorylation.

  • Denote that phosphorylation is the addition of a phosphate group to serine, threonine or tyrosine residues
    • Phosphorylation of serine and threonine to form phosphoserine and phosphothreonine is the most common amino acid modification in proteins.
  • Label amino acid 3 as serine and draw a P attached to it to represent its phosphorylation.
    • The size and negative charge of the phosphate group is thought to be why phosphorylation causes a slight change of shape of a protein.
  • Write that phosphorylation is a reversible modification, which generally induces a conformational change in a protein that changes its activity.
    • Because of this, phosphorylation is often used as a switch mechanism of protein activation.
    • Proteins are often phosphorylated (or dephosphorylated) in order to activate or deactivate them.
  • As a clinical correlate, write that epinephrine (adrenaline) changes enzyme activity via serine and threonine phosphorylation.
    • Many other hormones and neurotransmitters use phosphorylation to start signaling cascades and thus it has far-reaching implications for metabolism, immune response, and a range of other physiological functions.