Mutant Rat Trypsin
Andrew Worthington, '03
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+
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<beta-barrel structure<
There are four regions of alpha-helix<six disulfide bridges<
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,
IV. Mutant Trypsin
While rat (Rattus rattus) trypsin has thirteen
sites (12 shown) <Lys61-Ser62
peptide bond. In addition, the peptide
segment between these two sites <disulfidebridges<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+
the mechanism by which Ca2+
binding affects autolysis is still
unknown. Ca2+ has been shown to protect trypsin from autolysis.
The Ca2+-binding loop
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,
and Asp102, are
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.
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