Mutant Rat Trypsin

Andrew Worthington, '03

Biomolecules Index

I. Introduction

Trypsin is a proteolytic enzyme, important for the digestion of proteins. In humans, the protein is produced in its inactive form, trypsinogen, within the pancrease. Trypsinogen enters the small intestine, via the common bile duct, where it converted to active trypsin. Trypsin cleaves a terminal hexapeptide from trypsinogen to yield a single-chain [beta]-trypsin. Subsequent autolysis produces other active forms having two or more peptide chains. The two predominant forms of trypsin are [alpha]-trypsin, which has two peptide chains bound by disulfide bonds, and [beta]-trypsin.

Trypsin degrades proteins. As trypsin is itslf a protein, it is capable of digesting inself: a process called autolysis. Autolysis is important for the regulation of trypsin levels within living organisms. This regulation is assisted by Ca²⁺ions, which bind to trypsin (at the Ca²⁺-binding loop) and protect the molecule against autolysis. In living organisms, autolysis is controlled and normally does not cause problems. However, when working with trypin in vitro, the process of autolysis often poses some problems. For in vitro processes that require the use of trypsin, such as working with cell cultures or manufacturing insulin, trypsin's degradation can become expensive as active trypsin gets "used up." Developing mutant trypsin that does not auto-degrade could be of great use for researchers.

There are several sites on the trypsin molecule at which autolysis is known to occur. Research has been done to investigate these sites, because the inability of trypsin to self-degrade has been linked to human hereditary pancreatitis. This deadly disease is believed to occur due to inappropriate activation of trypsin within the pancrease. This results in the autodigestion of pancreatic tissue. In this investigation, we will be studying rat (Rattus rattus) trypsin and a mutant form of rat trypsin, in which two autolytic sites have been removed. This mutant form of trypsin could help researchers understand hereditary pancreatitis and could be useful for research that is dependent upon significant use of active trypsin.

II. General Structure

Trypsin is a globular protein of 24 kDa, composed of 220 residues.
The protein is composed of 13 beta-strands<>, six of which form a beta-barrel structure< >.
There are four regions of alpha-helix<>, and six disulfide bridges<>.
The Ca²⁺-binding loop <>extends from Glu70 to Glu80.

III. Catalytic Triad

The enzymatic activity of trypsin is highly specific towards the positive side-chains of residues lysine (Lys) and arginine (Arg), cleaving a peptide at the carboxyl side of these residues, during a hydrolytic reaction. The catalytic triad of trypsin forms the active site of the enzyme. Three amino acid residues, His57, Asp102, and Cys195<>, are vital to the proteolytic function of the molecule. In the absence of a substrate protein, His57 is unprotonated. However, when the sulfur atom of Cys195 carries out a nucleophilic attack on the substrate, His57 accepts a proton from Cys195. The role of Asp102 is to stabilize the positively charged form of His57 in the transition state. When the substrate moves in and binds to Cys195, a tetrahedral transition state is formed, with the substrate's carbonyl oxygen becoming negatively charged, as it forms a single bond. In the next step, the amine component of the substrate forms a hydrogen bond to His57, while the acid component is covalently bound to Cys195. The amine component diffuses away, thus completing the acylation stage of the hydrolytic reaction. During the next step, a water molecule takes the place of the amine component of the original substrate. Then His57 draws a proton away from water, while the resulting OH^- ion attacks the carbonyl carbon atom of the acyl group that is attached to Cys195 to form the next tetrahedral transition state. Finally, His57 donates the proton to the sulfur atom of Cys195, which then releases the acid component of the substrate.

IV. Mutant Trypsin

While rat (Rattus rattus) trypsin has thirteen potential trypsinsensitive sites (12 shown) <>, there are two especially important autolytic sites that have been reported: Lys61-Ser62 and Arg117-Val118<>. It is known that autolysis of wild-type trypsin begins with the cleavage of the Arg117-Val118 peptide bond. In addition, the peptide segment between these two sites <> is part of the longest peptide chain not stabilized by disulfidebridges<>; this region may function as a built-in target for autolysis. As long as both ends of the peptide remain intact, the other cleavagesites within this region appear to be protected from hydrolysis<>.

Site-directed mutagenesis of Lys61 and Arg117 to Asn61 and Asn117, resulted in a trypsin mutant that was almost completely resistant to autolysis. In addition, these mutations did not significantly alter the catalytic efficiency of the enzyme.

The rate of autolysis is dependent upon Ca²⁺concentration; the mechanism by which Ca²⁺binding affects autolysis is still unknown. Ca²⁺has been shown to protect trypsin from autolysis. The Ca²⁺-binding loop <>extends from Glu70 to Glu80, within the "self-destruction" segment. As the mutant trypsin was resistant to autolysis and does not need Ca²⁺ to protect it from autolysis, the protein was found to be almost completely insensitive to the presense of Ca²⁺. Finally, the loss of activity of wild-type trypsin can be explaned by the disruption of this peptide segment, as two members of the catalytic triad, His57 and Asp102, are near or a part of this region<>.

This double mutant trypsin may prove useful in experiments in which autolysis of wild-type trypsin has caused problems. Finally, this mutant trypsin will be useful in understanding and treating human hereditary pancreatitis.

V. References

Brookhaven Protein Data Bank (http://www.rcsb.org/pdb). PBD: 1DPO

Earnest, T., Fauman, E., Craik, C.S., Stroud, R.: 1.59 A Structure of Trypsin at 120K: Comparison of low temperature and room temperature structures. Proteins 10 pp. 171 (1991)

Ganassin, R.C., Bols, N.C.: Development of Long-term Rainbow Trout Spleen Cultures that are Haemopoitic and produce Dendritic Cells. Fish & Shellfish Immunology 6 pp. 17-34 (1996)

Stryer, Lubert. Biochemistry (New York: W.H. Freeman and Company, 1995), pp 56, 182, 222-227, 250-252.

Varallyay, E., Pal, G., Patthy, A., Szilagyi, L., Graf, L.: Two Mutations in Rat Trypsin Confer Resistance against Autolysis. Biochemical and Biophysical Research Communications 243 pp. 56-60 (1998)

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