Structural Biochemistry/Proteins/Proteolytic Analysis of Proteins

Overview
Proteolysis is a process of breaking down proteins into simpler compounds under the aid of proteases. This process takes place throughout the body for a variety of purposes. For example, food is digested into compounds that the body can use for cellular processes. Proteolysis can also link to some disease processes such as the venom causes tissue death by breaking down proteins of the person whom is bitten by a snake.

A protease is an enzymes that breaks downproteins through a process called proteolysis. Proteolysis breaks thepolypeptide bond that link amino acids together.

The selectivity in regulated proteolysis is controlled by generation and recognition of specific degrons on substrates.

Scientists have found that the N-end rule pathway is also a proteolytic system, in which N-terminal residues of short-lived proteins are recognized by recognition components (N-recognins) as components of degrons (called N-degrons). Substrates of N-recognins can be produced during the exposure of embedded destabilizing residues at the N terminus by protyolytic cleavage. In addition, N-degrons can also be produced through the modifications of posttranslationally exposed pro-N-degrons of stable proteins. The Modifications include oxidation, arginylation, leucylation, phenylalanylation, and acetylation. Proteolytic systems that base on generation and recognition of N-degrons have been seen in both prokaryots and eukaryotes. In general, the N-rule pathway regulates homeostasis of a number of physiological processes.

Proteases: types and functions
The proteases are categorized by the pH the enzymes work best in. There are acid proteases, neutral proteases, and basic proteases.

A protease's function is to cleave peptide bonds. They are enzymes that work best under acidic conditions. There actually many types of proteases; some examples include glutamic acid proteases and threonine proteases. Although most proteases operate under acidic conditions, there are also a few that operate under basic conditions, these are called alkaline proteases.





Proteases occur in all organisms. The cleaving action of a protease can either halt a protein's function or activate it. Proteases catalyze this peptide cleaving reaction by hydrolysis in which a water group acts as a nucleophile and attacks the peptide bond. Proteases can cleave other proteases. These enzymes play a huge role in digestion in that they cut proteins into fragments so the body can salvage and absorb the freed amino acids. The use of proteases in medicine is also popular as the study of proteases have helped us better understand inflammatory conditions and immune regulation. If a protein is part of a living cell and necessary for that cell to carry out interactions with other cells then proteases will not cleave it because normal living cells contain an inhibitor mechanism that stops the cleaving process. Protease deficiency can cause many health related problems. The acidity created in the stomach through protein digestion; if there are not enough proteases then this acidic equilibrium is disturbed causing an increase in alkaline character in the blood which could lead to insomnia or anxiety.

The study of protease structure has also been useful in providing a means for a structure-based drug design strategy to develop new products. For instance, HIV protease is an enzyme that is crucial to the development of HIV. Inhibiting this enzyme would help prevent the HIV virus from spreading throughout the body. By studying the structure of this enzyme, researchers hoped to determine types of molecules with the capability of blocking HIV protease. Thus giving us more insight into the effect that this disease has on the immune system so we can strive to minimize or eliminate the fatal consequences of fully-blown HIV. This strategy would prove faster and more efficient than the typical trial-and-error process, which could be lengthy and unsuccessful. (The Structures of Life, U.S. Department of Health and Human Services, http://www.nigms.nih.gov)

Generation of N-degrons by conjugation of amino acids
In both eukaryotes and prokaryotes (bacteria), conjugation of destabilizing amino acids to pro-N-degrons is the main way of producing primary destabilizing residues in the N-end rule pathway. This process is interceded by evolutionary conserved aminoacyl tRNA transferases, which allows pro-N-degrons to be recognized by N-recognins under certain conditions.

Arginylation in Eukaryotic N-rule pathway
In eukaryotes, the N-terminal Arg is structurally favored degron for the UBR box of N-recognins. The degron Arg can be induced by ATE1-encoded arginyl R-transferases. The Arg from Arg-tRNA is transferred to the N-terminal ɑ-amino group of acceptor subatrates. In mammalian, the ATE1 gene expresses at least six isoforms through alternative splicing of pre-mRNA. The importance of protein arginylation has been confirmed by the discorvery in which mouse embryos die because of defects in cardiac and vascular development due to ATE1 deficient.

Leucylation and phenyllanylation in prokaryotic N-end rule pathway
N-terminal Leu and Phe residues, primary destabilizing residues on bacterial proteins, can be induced by conjugation of destabilizing amino acids derived from aminoacyl tRNAs. Two types of aminoacyl transferases are found to be intercede leucylation and phenylalanylation in the N-end rule pathway.

Experiments have shown that the aat encoded Escherichia coli L/f transferase transfers Leu or Phe to the acceptors Arg and Lys (type 1 primary residues in eukaryotes).