Lecture 24 Summary

Protein degradation

Protein degradation occurs in the extracellular, catabolic process of digestion, and intracellularly by targeted degradation. The normal lifetimes of cellular proteins vary over a wide range. In bacteria and yeast, it has been demonstrated that the half-life of a protein is determined principally by the identity of its amino terminal residue - this is the so-called "N-end rule". In eukaryotes, targeted degradation occurs via the ubiquitin pathway. Targeted intracellular protein degradation offers the cell many regulatory possibilities.

Ubiquitin pathway

Ubiquitin (Ub) attachment requires the sequential action of three enzymes, E1, E2, and E3. Ubiquitin is activated in an ATP-dependent process by E1, resulting in the formation of a thioester between the C-terminal glycine of Ub and a cysteine residue of E1. Activated Ub is transferred to a cysteine residue of E2, a Ub-carrier protein. Ubiquitin is then ligated (via its C-terminus) to the target protein by a Ub-protein ligase, or E3, forming an isopeptide linkage.

The proteasome - a protein digestion machine

The proteasome is a macromolecular assemblage that acts as a ATP-dependent proteolytic machine within cells. The eukaryotic proteasome is a 26S (2000 kD) complex forming a hollow cylindical 20S (700 kD) assemblage, capped on either end by two 19S regulatory complexes. The 20S component is built up from four heptameric rings, two composed of a (alpha) subunits and two of b (beta) subunits, with the rings arranged in the order abba. While this basic architecture is conserved in organisms from archaea to mammals, eukaryotic 20S particles are composed of subtypes of a and b subunits. The 26S form seems so far to be limited to eukaryotes. The proteolytic activity of the proteasome resides in the b subunits, which have a threonine residue as the attacking nucleophile within a catalytic triad. The b subunits of the proteasome are thus members of the serine protease family of proteases.

Amino acid metabolism - a summary

Our major focus will be degradative (Ch.23) and synthetic (Ch.24) pathways of the amino acids, and how they relate to the main metabolic pathways we have already studied. Amino acids are not "stored" in the same sense as carbohydrates and fats as fuel reserves, so they are either built up into new polypeptides (or contribute in other ways to biosynthesis) or catabolized. The catabolism of amino acids is separable into two stages: deamination and metabolism of the remaining "carbon skeleton". Nitrogen from an excess of amino acids must be disposed of as a waste product. In humans, excess nitrogen is excreted as urea. The first step in nitrogen disposal is transamination, in which a-amino groups are transfered to a-ketoglutarate, forming glutamate and a new a-keto acid derived from the original amino acid. Glutamate is subsequently oxidatively deaminated. Carbon skeletons can be either glucogenic or ketogenic (or both).

Glutamate dehydrogenase (GDH): Key points

¶ Amino groups are funneled to glutamate by aminotransferases

¶ Oxidative deamination of glutamate is carried out by glutamate dehydrogenase (GDH) [EC 1.4.1.2 and EC 1.4.1.3].

¶ The reaction catalyzed by GDH is between glutamate plus NAD(P)+ and a-ketoglutarate, ammomnia, and NAD(P)H.

¶ The GDH mechanism involves reduction of the a-amino group of glutamate, yielding the intermediate a-iminoglutamate, which is then hydrolyzed to a-ketoglutarate and ammonia.

¶ The standard free energy of the reaction favors glutamate plus NAD(P)+, yet this enzyme is thought to act as a regulatory enzyme for amino acid catabolism.

¶ GDH is allosterically regulated: it is inhibited by GTP and NADH, and activated by ADP, leucine, and NAD+. Therefore a low energy charge stimulates GDH activity.

¶ Deregulated GDH activity is linked to hyperinsulinism-hyperammonemia (HI/HA) in humans.

Study questions

• Describe the regulation of protein turnover.
• Explain why the ubiquitination enzymes E1, E2, and E3 show increasing target specificity, in the order listed.
• Describe the structure and activity of the 20S catalytic core of the eukaryotic proteasome.
• Describe how targeted protein degradation is used to regulate the following biological processes:
  • Activation of pro-inflammatory response genes by NF-kB.
  • Antigen presentation by MHC Class II
  • Cell-cycle progression
• Distinguish between oxidative and non-oxidative deamination

Page updated 12-27-06

References

1. Berg, Tymoczko, and Stryer. Biochemistry (BTS): 6th edition (2007, Freeman) Ch.23, pp.649-661.
2. Pickart CM. (2001). "Mechanisms underlying ubiquitination". Ann Rev Biochem 70: 503-533.