ANEXO I: Conductividad Térmica de la Lana Cerámica.
TEMPEATURA PROMED ESTABILIZADA
8. CONCLUSIONES Y RECOMENDACIONES
Chymotrypsin is secreted into the gastrointestinal tract as an
inactive precursor, or zymogen, chymotrypsinogen.
Chymotrypsinogen has a single polypeptide chain of 245 residues held together by five intrachain d i s ulfide bridges.
Small amounts of trypsin slowly a c t i v a t e chymotrypsinogen into o-chymotrypsin. The latter undergoes a s l o w transformation into ß and y Chymotrypsins, /j-Chymotrypsin i s formed as a result of limited autolysis and >-chymotrypsin ari s e s as a result of a
pH-induced transition. A fast activation of a -
chymotrypsinogen yields two n e w forms o f Chymotrypsin, a very unstable form n , and a fairly stable f o r m 5 , which a r e more active than a Chymotrypsin *23 . The activa t i o n process depends
on the cleavage of the cyclic molecule o f a chymotrypsinogen by
trypsin, and b y Chymotrypsin itself (autolysis) (Fig. 1).
The activation of a chymotrypsinogen t o a Chymotrypsin is
triggered b y the tryptic cleavage o f the Arg-15 and Ile-16 bond 12/* . It appears that it is specif i c a l l y the formation of a new terminal amino group, rather than a n y "unblocking" reaction, which is responsible for promoting e n z y m i c activity * 23 . The
role o f the newly formed amino group o f I l e - 1 6 in stabilising the enzymatically active conformation o f o -Chymotrypsin h a s been
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investigated and it was shown that the negati v e l y charged 0 -
carboxylate group o f Asp-194 and the po s i t i v e l y charged a-amino group of Ile-16 form an ion-pair in a r e g i o n of presumably low dielectric constant.
Figure 1 : activation scheme for the Chymotrypsins.
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The overall 'tertiary structure o f the zymogen is similar to that of the active enzyme. There is no gross reorganisation in the
folding o f the main chain nor a significant helix-coil
transition.
The position of the chain termini created d u r i n g the activation
and subsequent autolysis are consistent w ith the cleavage of two dipeptides from the surface o f a zymogen but i n e a c h case leaving
the protein in the same conformation (Fig. 2).
Figure 2 : A view o f the complete p olypeptide chain of « - chymotrypsin (from Birktoft J.J. a n d B l o w D.M. *-2®).
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The stereochemistry of the activation o f t h e zymogen is
paralleled in the enzyme b y a pH-dependent structural transition. I n both transitions activity depends upon the integrity of the ion-pair between Ile-16 and Asp-194. A pH-dependent structure
transition occurs in the native enzyme between a n "active" and an "inactive" form. The active form dominates the equilibriun below p H 8 and is characterized functionally as the f o r m which binds
specific substrates in a productive mode 1 2 9 ^ The other
conformational state, the "inactive" form is f avoured at high p H and is unable to bind specific substrates and inhibitors in a
productive mode. This inability is considered to b e the basis for the fall-off in enzymatic activity at p H >8. S i n c e the optical rotation is different from that o f the active form, but similar to that of the zymogen, the inactive form is f e l t to resemble a-chymotrypsinogen in its tertiary structure.
The group with pKg 8 to 9 which controls this transition has been
identified as the a-amino group o f N-terminal Ile-16 By
treating the zymogen with acetic anhydride prior to activation, these workers prepared a derivative, acetyl-8-chymotrypsin, in which all amino groups except this one were acetylated. This
derivative was found to be fully active at neu t r a l pH, and to
undergo the transition to a n inactive f orm at high pH.
Reacetylation o f the derivative, so as to b l o c k the amino group, inactivated the enzyme.
This fact was latter contradicted b y P. Va l e n z u e l a and M.L.
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indicated that the deprotonation o f the isoleucine 16- amino group causes only a minor effect o n the binding a b i l i t y o f this enzyme. This led them to suggest that the peculiar behaviour of
a-chymotrypsin at alkaline p H may be caused b y the ionization of the phenolic group o f tyrosine 146 o r the amino g r o u p o f alanine
149, which are present as chain termini in a-chymotrypsin but
not i n 8 -Chymotrypsin. They investigated the preparation,
characterisation and kinetic properties at alkaline p H o f a new
stable and active form o f Chymotrypsin whi c h possesses threonine 147 instead of alanine 149 as the amino terminal g r o u p o f the C-
chain. This enzyme was called a j-chymotrypsin (Scheme 1).
Scheme 1: Schematic representation o f the structures of
chymotrypsinogen A (I), thr-neochymotrypsinogen (II),
and a^-chymotrypsin (III).
COON COON
I
II
COON
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They found that the kinetic properties of a^-chymotrypsin in
the neutral and alkaline pH regions strongly resemble those of 6-chymotrypsin. Their results strongly implicated the amino- terminus o f alanine 149 as a participant i n the reversible inactivation of a-chymotrypsin at high pH. Observations b y H.
Kaplan and H. Dugas seem to confirm the postulated
involvement o f alanine 149. Studying the properties o f the
isoleucine-16 amino group b y a competitive labeling technique,
Kaplan found that above p H 9.8, a-chymotrypsin undergoes an
additional conformational change not controlled b y the isoleucine
16 terminus. Furthermore, experiments with the alanine-149 amino group suggested that this group is involved in a conformational change o n deprotonation o f the isoleucine-16 a m i n o terminus.
Despite these results, it seems that the active conformation is stabilized by the ion-pair formed by Asp-194 a n d Ile-16. The existence o f the ion-pair accounts for the anomalously high pKa o f the a amino group o f Ile-16, b y comparison w i t h the pKa of less than 8 which is usual for peptide a a m ino groups. If the positive charge is removed from the amino group, the high potential created b y a negative charge in a medium o f low
dielectric constant within the molecule w o u l d require the
carboxylate ion to seek a n alternative orientation *n which
it would point into the solvent. The model suggests that this
could be accomplished b y a movement of the carboxylate group into the vicinity of the side group o f Ser-195 and His-57. Although the exact nature of the disruption of the active site cannot be
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predicted, it is suggested that at high p H the conformation in the region o f the active centre reverts to that o f the zymogen.
This could account for the absence of enzymatic activity in both proteins.
It is against that background that w e found it would be
interesting to investigate the behaviour o f the different forms of chymotrypsin and their ability to catalyze peptide synthesis
w h e n suspended in organic solvent.
Peptide bond formation involves the two subsites S and S' ^ of chyrootrypsin, and therefore would give m ore information about the tridimensional structure o f the enzyme than studies o f hydrolytic reactions i n which only the S-subsite is concerned. The ability
o f synthesizing peptide bonds b y the different forms of
chymotrypsin could help to provide a valuable insight into molecular recognition, and a study o f the behaviour of these enzymes in organic solvents could yield information about the
intimate effect o f the new environment on the molecular
structure.
The ability o f the different forms of chymotrypsin to synthesize
peptides in organic media was determined b y estimating the amount o f condensation products formed during the reaction between N-
acetyl-L-tyrosine ethyl ester and L-phenylalaninamide in
dichlorome thane.
The experimental procedure was the following : to a solution of
N-acetyl-L-tyrosine ethyl ester and L-phenylalaninaraide (40mM,
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form of chymotrypsin. To the remaining suspension was then added 0 .2% (v/v) o f water. The suspension was t hen stirred 1 hour at r o o m temperature, filtered, a n d the residue w a s washed w i t h warm
methanol. After evaporation o f the solvents, the content o f the reaction mixture was analysed b y *H NMR (220MHz, CDCI3/TFA 1:1). T h e results are depicted in Fig. 3.