701
Biochem. J. (1976) 153, 701-712
Printed in Great Britain
Effects of Secondary Binding by Activator and Inhibitor Peptides
on Covalent Intermediates of Pig Pepsin
By TUSN-T. WANG and THEO HOFMANN
Department ofBiochemistry, University of Toronto, Toronto M5S 1A8, Canada
(Received 15 September 1975)
A number of peptides were found to increase the activity of pig pepsin towards small
synthetic substrates. The activators increase transpeptidation of both the acyl-transfer
and the amino-transfer types by as much as 45-fold. The effect on hydrolysis varies from
inhibition to modest activation, but is always less than the effect on transpeptidation. The
kinetics of substrate cleavage are the converse of non-competitive inhibition and show an
increase in kca,. and no effect on Km values. Lineweaver-Burk plots of results obtained
in the presence of the activators indicate a substrate activation at high substrate concentration. This appears to be a co-operative effect, since it is not observed in the absence of the
activators. The activation is greatest at pH4.7, less at pH3.4, and at pH 2.0 is observable
only with some of the activator peptides. The results show directly the effect of secondary
binding on the catalytic efficiency of pepsin. The most effective activators are those that
are most hydrophobic. The results suggest that binding in the secondary binding sites
causes an increase in hydrophobicity in the catalytic site which results in increased stability
of the acyl and amino intermediates, and preferential reaction with acceptors other than
water. The implication that the present results strengthen the case for a role of covalent
intermediates in the hydrolysis of good substrates (high kcat. values) is discussed.
The specificity of pig pepsin is determined not only
by the amino acid residues involved in the sensitive
bond but also by amino acid residues more distant
from the catalytic point (Fruton, 1970; Clement,
1973). In particular, the interaction of an extended
substrate chain in a secondary binding site causes
large increases in the catalytic efficiency that are
mainly due to increases in kca,t. (Fruton, 1970). At
the most, only small changes in Km values are observed. These 'secondary interactions', are also
important for the pepsin homologues, chymosin,
cathepsin D (Sampath-Kumar & Fruton, 1974),
penicillopepsin (Wang et al., 1974), Rhizopus pepsin,
and other microbial proteinases (Oka & Morihara,
1973). Significant but less pronounced effects of
secondary interactions are also observed with other
proteinases such as papain (Schechter & Berger,
1967), subtilisin (Morihara et al., 1971; Ottesen et al.,
1969), carboxypeptidase A (Schechter & Zazepizki,
1971) and elastase (Gertler & Hofmann, 1970;
Thompson & Blout, 1970).
Fruton (1970) suggested that the effect of the
secondary interactions with pepsin was due to a
conformational change which brings about the
increase in catalytic efficiency. Recently we obtained
direct evidence from circular-dichroism spectra for a
conformational change associated with binding of
Leu-Gly-Leu to penicillopepsin (Wang et al., 1974).
Leu-Gly-Leu is not a substrate but activates the
Vol. 153
cleavage of a substrate with an activation constant
that has about the same value as the binding constant
determined from the circular-dichroism changes
(Wang et al., 1974). The observation that nonsubstrates can activate the cleavage* of substrates by
increasing kcat. but not Km has now been extended to
pig pepsin.
The present study describes the effects of a variety
of peptides (which will be called 'modifiers') on the
cleavage of substrate peptides. Kinetic studies were
carried out to determine the effects of the modifiers
on kca,., Km and the ratios hydrolysis/transpeptidation
of both the acyl- and amino-transfer types.
Experimental
Materials
Crystalline pig pepsin (lot 300001) was purchased
from Calbiochem, Los Angeles, Calif., U.S.A.; pepsin
was also prepared from pepsinogen (Worthington
Biochemical Corp., Freehold, N.J., U.S.A.) by the
procedure of Rajagopalan et al. (1966). All other
chemicals were described in the preceding paper
(Wang & Hofmann, 1976).
* The term
'cleavage' is used in the present paper to
imply that the reaction is either a hydrolysis or a transpeptidation, or both. The term 'hydrolysis' will only be
used for strictly hydrolytic reactions.
702
Methods
Generalprocedures for transpeptidation and hydrolysis studies. These were described in the preceding
paper (Wang & Hofmann, 1976).
Peptide isolation and identification. In an experiment in which Cbz*-Leu-Met and Leu-Trp-Met-Arg
were incubated together, it was necessary to isolate
the products of the reaction for identification and
characterization. In this case the reaction was carried
out in 0.05M-acetic acid buffer, pH3.4, instead of
sodium citrate buffer. The reaction product was a
peptide that had precipitated from the incubation
mixture and had no free N-terminal. It was presumed
to be a benzyloxycarbonyl derivative. To remove the
blocking group, the precipitate was dissolved in
0.2ml of acetic acid saturated with HBr. The mixture
was incubated for 30min at 22°C. The solution was
evaporated in vacuo and the residue dissolved in the
high-voltage electrophoresis buffer (pyridine/acetic
acid/water; 1:10:89, by vol.). The free peptide was
purified by high-voltage electrophoresis at pH 1.9
and 3.6 in the volatile buffers described by Bennett
(1967). Guide strips from the electrophoretograms
were stained with cadmium/ninhydrin reagent of
Heilmann et al. (1957). The peptidewaselutedfrom
the unstained paper with a few drops of the pH 1.9
high-voltage electrophoresis buffer. Amino acid
analysis was carried out after hydrolysis with
0.2m1 of constant-boiling HCI for 20h at 110°C in
evacuated sealed tubes on a Beckman 121C amino
acid analyser. The dansyl-Edman technique decribed
by Gray (1967a,b) was used for the determination of
the N-terminal groups and the sequence of the pep.
tide. The dansyl amino acids were identified on
polyamide sheets (5cm x 5cm) (Ching-Chen Trading
Co., Taipei, Taiwan) as described by Hartley
(1970).
The time-course experiments described in the
preceding paper (Wang & Hofmann, 1976) show that
the progress curves for the formation of most of the
products are not linear and therefore measurement of
initial rates is not possible for many of the reactions.
In the kinetic experiments described in the present
paper apparent rates are based on samples taken only
at definite times given in the legends to the Tables and
Figures. Depending on the nature of the reaction
products these apparent rates are either higher or
lower than initial rates, depending on whether the
progress curve is sigrnoidal [as for the formation of
Leu-Leu-Leu from Cbz-Phe-Leu (Wang & Hofmann,
1976)] or concave towards the abscissa respectively.
Where evidence for linearity of the release of a
product was available, the rate is given as apparent
jxitial rate and indicated in the legends to the Figures.
This is the case,, for instance, with the release of
Tyr,NH2 from Leu-Tyr-NH2 by pepsin (Takahashi
* Abbreviation: Cbz, benzyloxycarbonyl.
T.-T. WANG AND T. HOFMANN
et al., 1974). However, even in this case the observed
linearity represents only an apparent initial rate
because Tyr-NH2 is released not only from the
substrate Leu-Tyr-NH2 but also from the putative
intermediates Leu-Leu-Tyr-NH2 and Leu-Leu-LeuTyr-NH2. The rates of cleavage, of the latter two are
comparable to that of the original substrate (see
Legend to Fig. 1) and therefore not negligible. Little
deviation was observed from linearity of release of
Tyr-NH2 from Leu-Tyr-NH2 in the presence of
Cbz-Leu-Met (4mM) or Leu-Gly-Leu (6mM) over the
time period used in the experiments. It is clear,
however, that in all experiments, where relevant,
kinetic values K1, Ka and kcat. are apparent and
complex parameters.
Results
In the experiments described below, the effects of a
number of peptides that modify the activity of pepsin
acting on other small peptides are studied. Although
in most cases the modifying peptides increased the
activity, inhibition was observed under certain
conditions. For this reason those peptides that are
not substrates or were not studied as substrates in a
particular experiment will be called 'modifier' peptides. The modifier peptides investigated are either
resistant to cleavage by pepsin or, if cleaved, are
cleaved more slowly than the 'substrates used in the
same experiment.
Activation ofacyl transfer
The effect of Cbz-Leu-Met on the hydrolysis and
transpeptidation reactions of Leu-Tyr-NHz at
pH4.7 is shown in Fig. 1. The ratio of initial rates of
cleavage of the substrate or formation of transpeptidation products at various modifier concentrations to
the initial rates in its absence are plotted against the
modifier concentration. Although the rates of formation of all products are increased, the largest effect is
observed on the formation of Leu-Leu-Leu and the
smallest on the product of hydrolysis, leucine. For the
transpeptidation, optimum activation is seen at
approx. 6mM-Cbz-Leu-Met but hydrolysis increases
up to lomM. The modifier peptide also acts as a
substrate as seen by the formation ofthe transpeptidation products Met-Met or Met-Met-Met and
methionine. Fig. 2 shows the even more pronounced
effect of Cbz-Leu-Leu on the cleavage of Leu-TyrNH2. The cleavage, as measured by the liberation of
Tyr-NH2, increases some 24-fold at a concentration
of the modifier about 6mM. The increase in the transpeptidation product, however, is not solely due to the
cleavage of the substrate but presumably also receives
a contribution from the cleavage of the modifier.
Further, the reactions proceeding in this system are
1976
EFFECTS OF NON-SUBSTRATE PEPTIDES ON PIG PEPSIN
probably complicated by the presence of a condensa.
tion reaction between the modifier and the substrate,
as shown by the formnation of some insoluble product
on prolonged reaction and high concentration of the
modifier. The insoluble product is formed only at
concentrations of Cbz-<Leu-Leu above 61UM. Its
formation may well be responsible for the decrease
in the activation at the higher modifier concentrations.
In spite of the side reactions it is clear that Cbz-LeuLeu is a more effective activator than Cbz-Leu-Met
703
(Fig. 1) as shown by the increased rate of formation
of Tyr.NH2 which is due only to the cleavage of the
'substrate'. The activation of the cleavage of Leu-Tyr.
NH2 by Cbz-Leu"Leu is the result of several effects,
some of which oppose each other. The kinetic parameters for the system are shown in Table 1. If treated
as simple, non-competitive activation (Wang et al.,
80
70
lo
0
|0
-
60
8
50
6
R40
I
30
I
20
0
10
L
0
2
4
6
8
10
[Cbz-Leu-Met] (mM)
Fig. 1. Effect of Cbz-Leu-Met on cleavage ofLeu-Tyr-NH2
(2mM) at pH4.7, 370C,for 3h
[Pepsin] = 0.6mg/ml. va/vO was calculated from the
apparent rates of product formation in the absence (vo)
and presence (va) of Cbz-Leu-Met. vo values (aM. min-'):
2.2x 10-1 (Tyr-NH2); 3.3 x 10- (leucine); 2.7x 102
(Leu-Leu); 3.3 x 10-2 (Leu-Leu-Leu); o, leucine; 0,
Leu-Leu; 0, Leu-Leu-Leu; A, Tyr-NH2.
2
4
6
8
[Cbz-Leu-Leu] (mM)
Fig. 2. Effect of Cbz-LeauLeu on cleavage ofLeu-Tyr-NH2
(2mM) at pf4.7, 37°C,for 3 h
Va/Vo was determined as for Fig. 1; vD values were as given
in Fig. 1. [Pepsin]= 0.6mg/mi. CJ, Leu-Leu-Leu; 0,
Leu-Leu; o, leucine; A, Tyr-NH2.
Table 1. Kinetic parameters for the cleavages ofLeu-Tyr-NH2 in the presence and absence of modifiers
The rate of formation of Tyr-NH2 was measured after a 5h incubation in the absence of modifiers and 3 h incubation in
thepresence of modifiers. Kawascalculatedas described by Wang etal. (1974). [Substrate] = 0.2-4mM; [pepsin] = 0.6mg/ml.
Because of the complexity of the reactions Km. K. and kcat. are apparent and complex parameters, as discussed in the
Experimental section.
Modifiers
Ka of modifiers
103 X k,at.IK.,
pH
Km
(concn. in reaction mixture)
103 x kcat. (s-1)
(mM)
3.4
Substrate
Leu-Tyr-NH2 only
5mM
2.2
0.4
+Met-Gly-Met-Met (2mr)
5mM
8.9
1.8
2.7+0.1
+Met-Gly-Met-Met (8mM)
5mM
14.9
3.0
4.7
4.7
Vol. 153
Substrate
Leu-Tyr-NH2 only
+Leu-Gly-Leu (2mm)
+Leu-Gly-Leu (6mm)
Substrate
Leu-Tyr-NH2 only
+Cbz-Leu-Leu (2 mM)
+Cbz-Leu-Leu (6mM)
4mM
4mM
4mM
4mM
4mM
4mM
0.8
1.2
1.9
0.2
0.3
0.48
Over 20
0.8
5.2
6.7
0.2
1.3
1.7
0.73 + 0.2
T.-T. WANG AND T. HOFMANN
704
1974) the activation constant for Cbz-Leu-Leu is
0.73mM, and the Km for the substrate is 4mM and is
not affected by the modifier. In the experiments
shown in Fig. 2, the substrate is well below saturating
conditions and the effect of the modifier is most
noticeable at a concentration that is almost ten times
that of the activation constant. The results presented
in Fig. 2 are complex. In addition to a direct activation
effect by the modifier, there may also be effects of
activation due to Leu-Leu-Leu. In a separate experiment it was shown that 2mM-Leu-Leu-Leu added at
the beginning increased 22-fold the cleavage of LeuTyr-NH2 (2mM) as measured by the release of
Tyr-NH2 after 3 h. Further, the modifier peptide can
also be expected to exert an inhibitory effect since it
has been shown to act as a substrate (Wang &
Hofmann, 1976). However, the inhibitory effect is
obviously outweighed by the activation effects. The
complexity is increased by a probable co-operativeactivation effect between modifier and substrate, evidence for which is shown in Figs. 3(a) and 3(b). When
Leu-Gly-Leu is used as an activator, the LineweaverBurk plots (Fig. 3a) are no longer linear but show
downward curvatures at high substrate concentrations. The most probable explanation is that there is
substrate activation. This, however, can only be
observed in the presence of the modifier and not in
its absence. This is more clearly shown in a plot of
(S]/v against [S] (Fig. 3b). Similar effects are detectable when Cbz-Leu-Leu is used as a modifier (Figs.
3a and 3b).
The effect of pH on the modifying effect of CbzLeu-Leu on the substrate Leu-Tyr-NH2 is shown in
Fig. 4. In this case only initial rates of formation of
tyrosine amide were measured. The results show quite
clearly that the highest degree of activation is
observed at pH4.7 whereas at pH3.4 the maximum
activation is only about one-quarter of that at pH4.7
and no activation is observed at pH2. As shown
previously (Wang & Hofmann, 1976) Leu-Tyr-NH2
is hydrolysed only very slowly at pH2, where no
transpeptidation is observed. Fig. 5 shows the effect
of two other modifiers, Cbz-Leu-Met and Leu-GlyLeu at pH4.7, where again marked activation is
observed, and at pH3.4, where the activation by
Cbz-Leu-Met is hardly noticeable. Activation by
Leu-Gly-Leu at pH3.4 only becomes apparent at
10mM. Fig. 6 shows that another modifier, Met-GlyMet-Met, shows considerable activation at pH3.4. In
this case the major activating effect is on the formation
of the dipeptide Leu-Leu (up to 45-fold at 6mM
modifier), and the tripeptide formation Len-Leu-Leu
is increased to 15-fold and the total cleavage some fivefold (Fig. 6b). The increase in the rate of formation of
leucine, the product of hydrolysis, is very low. Fig.
6(a) also shows the effect of some related tetrapeptides
and of the tripeptide Glu-Gly-Phe. These four peptides cause little or no activation. The kinetic analysis
in Table 1 shows that the activation constant for
Met-Gly-Met-Met is 2.7mM. This activating effect by
Met-Gly-Met-Met is especially interesting since
Met-Gly-Met-Met is the only modifier we used in this
series of experiments that was also a good substrate
(as judged from the rate of release of methionine).
In the preceding paper (Wang & Hofmann, 1976)
we showed that the best substrate for the study of
E-4
1-1
10-3 X 1/[S] (M-1)
-4
-3
-2
-I
0
1
2
3
4
103 X [S] (M)
Fig. 3. Non-competitive activation of cleavage of Leu-TyrNH2 (as measured by release of Tyr-NH2) by Leu-Gly-Leu
(3 h) and Cbz-Leu-Leu (1 h) respectively (pH4.7, 37'C)
[Pepsin] = 0.6mg/ml. Apparent initial rates of release of
Tyr-NH2 were observed (see comments in the Experimental section). o, Leu-Tyr-NH2 alone; with Leu-GlyLeu: A, 2mM; A, 6mM; with Cbz-Leu-Leu: *, 2mm; *,
6mM. (a) Lineweaver-Burk plot; (b) same results as plot of
[S]/v against [S].
1976
70S
EFFECTS OF NON-SUBSTRATE PEPTIDES ON PIG PEPSIN
10
a
8
o
2
4
6
8
10
0
2
4-
4
6
8
l0
[Modifier] (mM) [Met-Gly-Met-Met] (mM)
~~~~A
_
0
2
0
4
10
8
6
[Cbz-Leu-Leu] (mM)
Fig. 4. Effect of Cbz-Leu-Leu on cleavage ofLeu-Tyr-NH2
(2mM) at different pH values and at 370C
[Pepsin]= 0.6mg/ml. va/vO is the ratio of the apparent
(v.) and without (vo)
rates of release of Tyr-NH2 with
Cbz-Leu-Leu. vo values (aM min-'): 2.2x 10-1 at pH4.7;
7.Ox 10-1 at pH3.4; 1.lx10-2 at pH12.0. o, pH4.7, 3h;
El, pH3.4, 3h;
pH2.0, 5h.
Fig. 6. Effect of various modifiers on cleavage ofLeu-TyrNH2 (2mM) at pH3.4 (37°C, 3 h)
[Pepsin] = 0.6mg/ml. v./vO is the ratio of apparent rates of
release of products with (v.) and without (vo) mnodifiers.
(a) Various modifiers; product Tyr-NH2; vo = 0.7pMmin-'. o, Met-Gly-Met-Met;
acetyl-Gly-Gly-Met;
A, Gly-Leu-Gly-Leu; El, Phe-Gly-Gly-Phe; *, Glu-GlyPhe. (b) Modifier: Met-Gly-Met-Met. Products: A, TyrNH2, v0=0.7pM/min; o, leucine, vO=0.12,M min-1;
0, Leu-Leu, vo = 3.3 x 10-2gM min-'; El, Leu-Leu-Leu,
vo = 4.7 x 10-2.M *min7l.
U,
A,
I
10
(a)
- (b)
8
6
0
4
2
0
I
2
I
4 6 8 10
[Cbz-Leu-MetJ (mM)
0
I
I
I
l0
[Leu-Gly-LeuJ (mM)
0
2
4
6
8
Fig. 5. Effect of Cbz-Leu-Met (a) and Leu-Gly-Leu (b) on
cleavage ofLeu-Tyr-NH2 (2mM) (37°C, 3h)
[Pepsin] = 0.6mg/ml. v,IvO is the ratio of the apparent
rates of release of Tyr-NH2 with (va) and without (vo)
modifiers. vo values (uMmmin-1): 2.2x1O-1 at pH4.7;
7.Ox 10-1 at pH3.4. o, pH4.7; El, pH3.4.
Vol. 153
acyl-transfer reactions so far found is Leu-Trp-MetArg. Fig. 7 shows the effect of Cbz-Leu-Met on both
hydrolysis and transpeptidation of this tetrapeptide
at pH3.4. The cleavage of Leu-Trp-Met-Arg was also
followed spectrophotometrically by measuring the
decrease in E294. Difference spectra between substrate
and substrate-with-pepsin recorded as a function of
time showed the appearance of a negative peak at
294nm. This is due to the protonation of the a-amino
group of tryptophan. Although we could not
measure the difference in molar extinction between
substrate and product because on prolonged incubation pepsin further cleaves Trp-Met-Arg, an approximate value of 600 litre- mol-- cm-' can be estimated
from the difference in molar extinction between the
protonated forms ofglycyltryptophan and tryptophan
(Donovan et al., 1961). After incubation for 10min the
reaction was stopped by boiling the mixture, and the
formation of the products Leu-Leu-Leu, Leu-Leu and
leucine was determined as described by Wang &
Hofmann (1976). Under these initial-rate conditions
very little Leu-Leu is formed. Its formation appears
to be unaffected by the modifier and is not shown in
Fig. 7. Leu-Leu is probably the product of hydrolysis
of Leu-Leu-Leu. Fig. 7 shows clearly that the most
pronounced activation (approximately ninefold) is
observed for the formation of Leu-Leu-Leu at a
concentration of the modifier between 1 and 1.5mM.
2A
706
-0
10
C
l1
0
0.5
1.0
1.5
2.0
2.5
[Cbz-Leu-MetJ (mM)
Fig. 7. Effect of Cbz-Leu-Met on cleavage ofL.eu.Trp-MetArg (1 mM) at pH3.4 (250C, l0mm- 1)
[Pepsin] = 0.6mg/ml. va/vo is the ratio of t he apparent
rates with (v.) and without (vo) modifiers vo values:
AE294,= 1.3x 10-3 litre mol-l cml-min ; leucine,
0.6pM nminl1; Leu-LeouLeu, 0.9#M'min-1. o, leucine;
0,LOu-Le-Leuu A, E294.
T12
t,10
X
6
-
4
0
-
3 2
u
0. 5
1.0
1.5
2.0
2.5
[Cbz-Leu-Met] (mM)
Fig. 8. EDfect of Cbz-Lea-Met on formation of
from Leu-Trp-Met-Arg (1 mm) asfunction ofpH
pH at
at 370C
37°C
[Pepsin] - .6mg/ml. Incubation time pO43.4, 4min;
pH2.0, 30min.
[I, pH3.4; A,'pHi2.0.
There is only about a 2.5-fold increase in the
bydrolysis. Since the increase in the cldavage of the
Leu-Trp bond, as shown by the changing absorbance,
is very close to the increase in transpeptidation, it 'is
clear that at all points the transpeptidation reaction
T.-T. WANG AND T. HOFMANN
Table 2. Activation of cleavage of Met-Tyr-Phe-NH2
(2mM) by various modifiers
va/vO is the ratio of pparent rates of product formation by
pepsin (0.6mg/i) in the absence (vo) and presence (va) of
modifier (2mA) at pH4.7. The reaction nmixture was
incubated for 20min in 0.05M-sodium citrate buffer,
pH4.7, at 370C. Since Met-Met and Met-Met-Mlet cannot
be separated completely, the Met-Met found may contain
some Met-Met-Met. A trace of Met-Tyr (<3nmol) was
also present, but was not affected by the modifiers.
vo values (#uM min11): 0.54 (Met); 0.61 (Met-Met).
Modifiers (2mM)
va/vo (Met)
Va/vo (Met-Met)
0.8
26.4
Cbz-Leu-Leu
0.9
Leu-Gly-Leu
3.5
1.6
Leu-Leu
0.9
Phe-Gly-Gly-Phe
0.8
2.1
Ala-Ala-Ala-Ala
1.4
is the major reaction. At a concentration of the modifier greater than 2mM precipitation occurred in the
reaction nixture. In an experiment carried out at a
concentration of Cbz-Leu-Met of 2.5mM at room
temperature (22°C) for 15min this precipitate was
centrifuged down and analysed as described in the
Experimental section. After removal of the benzyloxy-
carbonyl group, the tripeptide Leu-Met-Leu was
unambiguously identified. This, is presumably a
product of an initial condensation reaction between
the modifier and the substrate, followed by cleavage
at a Leu-Trp bond.
It was shown previously that Leu-Trp-Met-Argis
a very poor substrate at pH2; only small amounts of
leucine and Leu-Leu are formed whereas Leu-LeuLeu was undetectable (Wang & Hofmann, 1976).
The presence of the modifier Cbz-Leu-Met, however,
leads to significant formation of Leu-Leu-Leu
(Fig. 8). The modifier had no effect on leucine and
Leu-Leu formation. The effectiveness ofCbz-Leu-Leu
as modifier of acyl transfer (Fig. 2) is also shown in
Table 2 where the substrate is Met-Tyr-Phe-NH2.
The major products of cleavage of this substrate are
Met-Met and Met-Met-Met. Only small amounts of
free methionine are formed. None of the modifiers
affects methionine formation, but they increase the
transpeptidation reaction. Although Cbz-Leu-Leu
is a very effective activator, Leu-Leu has only a little
influence. The requirement for hydrophobic side
chains is apparent from the different effects of
Leu-Qly-Leu and Cbz-Leu-Leu on one hand and
Ala-Ala-Ala-Ala on the other.
Activation of amino transfer
In the preceding paper (Wang & Hofmann, 1976)
it was shown that Cbz-Phe-Leu at pH4.7 is a good
substrate for the demonstration of the transpeptidation of the amino-transfer type. This transpeptidation
1976
707
EFFECTS OF NON-SUBSTRATE PEPTIDES ON PIG PEPSIN
0
o
2
4
6
8
10
0
2
4
[Modifier] (mM)
Fig. 9. Effect ofmodifiers on cleavage of Cbz-Phe-Leu (2mM) at pH4.7 (37°C, 45min)
[Pepsin] = 1.6mg/ml. v./vo is the ratio of apparent rates of product formation with (v.) and without (vo) modifiers.
vo- (M minn) -0.4 for leucine; 1.5 for Leu-Leu-Leub Insufficient Leu-Leu was formed for quantification. o, leucine;
O, Leu-Leu-Leu. Modifiers: (a) Tyr-Leu-NHI; (b) TYr-Phe-NH2; (c) Tyr-Tyr-NH2.
Table 3. Activation of cleavage of Gly-Phe-Phe (2mM) by
various modifiers
v./vO is the ratio of initial rates of formation of Gly-Phe by
0.6mg of pepsin/ml in the absence (vo) and presence (va) of
modifier (2mM, except as otherwise specified). The
reaction mixture was incubated for 20min in (.OSMsodium citrate/HCI buffer, pH3.4 or 4.7, at 370C. vo values
(JLM min-'): 1.8 at pH3.4; 1.1 at pH4.7.
Modifiers
vI/vo at pH 3.4 v1/vo at pH4.7
0.9
Cbz-Leu-Leu
0.33
Tetra-Ala
1.0
1.0
Leu-Leu-NH2
Leu-Tyr-NH2
Ala-Tyr-Phe-NH2
Tyr-Leu-NH2
Tyr-Phe-NH2
Leu-NH2 (8mM)
Tyr-NH2 (8mM)
2.2
2.8
2.6
4.2
5.6
3.2
5.7
3.4
5.7
7.1
1.0
1.1
reaction can also be modified by added peptides, as
was shown by the effects of three modifiers, Tyr-LeuNH2, Tyr-Phe-NH2 and Tyr-Tyr-NH2 (Fig. 9). With
modifier concentrations up to 2mM the activation of
transpeptidation is between 2.5- and 3.5-fold. At
higher concentrations, however, the activation
decreases. Inhibition is observed with Tyr-Leu-NH2
at concentrations higher than 6mM. The hydrolysis
of the substrate shows no activation with Tyr-LeuNH2, a slight activation at ImM with the other two
Vol. 153
modifiers, but at higher concentrations a considerable
inhibition is observed. Tyr-Leu-NH2 inhibits the
hydrolysis by as much as 90%. As we have shown
(Wang & Hofmann, 1976) the initial rate of Leu-LeuLeu formation is about five times that of leucine
formation at pH4.7. The only modifier that inhibits
the overall cleavage is Tyr-Leu-NH2 at concentrations
higher than 6mM.
Further studies on the effect of modifiers on
substrates in which the C-terminal amino acid is
cleaved were carried out with Gly-Phe-Phe and with a
preparation of pepsin that was prepared from
pepsinogen by the method of Rajagopalan et al.
(1966). Table 3 shows the effect of a number of
modifiers on the cleavage of Gly-Phe-Phe at pH4.7
and 3.4. The cleavage was determined by measuring
the rate of formation of Gly-Phe. One of the most
noteworthy findings is that CbzLet-Leu acts as an
inhibitor of the reaction, especially at pH3.4. This
is in strong contrast with the activating effect which
this modifier has on the substrate that show acyltransfer reactions and indicates the strong affinity
of this peptide for the secondary site A as defined by
Takahashi & Hofmann (1975). The best activators
were found to be Tyr-Leu-NH2, Tyr-Phe-NH2,
Ala-Tyr-Phe-NH2, Leu-Tyr-NH2 and Leu-Leu-NH2.
The latter two are also substrates of the acyl-transfer
type as shown above. No effect was observed by
Leu-NH2, Tyr-NH2 and tetra-alanine.
The effect of pH on the cleavage of Gly-Phe-Phe
in the presence of three modifiers is shown in Figs.
T-T. WANG AND T. HOFMANN
708
I0
(a)
(c)
8
6
4
2
0
2
4
6
8
10
0
2
4
6
8
10
0
2
4
6
8
10
[Modifier] (mM)
Fig. 10. Effect of modifiers on cleavage of Gly-Phe-Phe (1 mM) at different pH values (370C, 30min)
[Pepsin] 0.6mg/ml; v./vO is the ratio of apparent rates of formation of Gly-Phe with (v,) and without (vo) modifiers.
vo values (#M-min-'): 1.1 at pH4.7; 1.8 at pH3.4 and 2.0; 0, pH4.7; [1, pH3.4; A, pH2.0. (a) Tyr-Leu-NH2; (b) Tyr-PheNH2; (c) Leu-Leu-NH2.
=
Table 4. Effect ofpHandmodifiers on kinetic parameters ofcleavage of Gly-Phe-Phe as measured by release ofGly-Phe at 37°C
K. was calculated as described by Wang et al. (1974). [Substrate] = 0.2-1.0 mM; [pepsin] = 0.6mg/ml. Reaction time 30min.
Because of the complexity of the reactions Km. Ka and k,.t. are apparent and complex parameters, as discussed in the
Experimental section.
Modifier
pH
Km (1M) 103xkcat. (s-1) 103xkcat./Km (mM"-s-1)
K. (mM)
1.3
Substrate only
1.0
2.2
2.2
2.0
Substrate only
0.9
2.1
2.5
0.9
+1 mM-Tyr-Leu-NH2
3.9
4.3
0.65
0.9
6.2
6.9
+6mM-Tyr-Leu-NH2
2.7
Substrate only
1.0
3.1
3.1
Substrate only
3.4
0.8
2.6
3.3
0.8
+1 mm-Tyr-Leu-NH2
10.5
8.4
0.42
0.8
+6mM-Tyr-Leu-NH2
16.8
13.2
4.0
Substrate only
1.1
3.3
3.0
4.7
Substrate only
1.4
1.9
1.4
1.4
10.3
+1 mM-Tyr-Leu-NH2
7.4
0.16
1.4
+6mM-Tyr-Leu-NH2
11.6
8.3
10(a), 10(b) and 10(c). As was found with the acyltransfer substrates (Figs. 4 and 5) the most pronounced activation was obtained at pH4.7, somewhat
lower activation at pH3.4 and the lowest activation
at pH2. With Leu-Leu-NH2 no activation was
observed. The effect of the modifier Tyr-Leu-NH2 on
the cleavage of Gly-Phe-Phe as a function of pH is
shown in Table 4 where the kinetic parameters kca,.
and Km are shown. The Michaelis-Menten constant
is not affected either by pH or by the presence of the
modifier as was found for the other substrates
described above. The pH-dependence of kcat. in the
absence of modifier shows a very shallow curve and
the specificity constant, kcat.IKm, shows an optimum
at pH3.4. In spite of the higher degree of activation
at pH4.7, at a modifier concentration of 6mM the
pH optimum of the reaction remains at pH3.4. The
definite activation at pH2 contrasts with the absence
of activating effects observable with the acyl-transfer
substrates (Fig. 4). Some of the Lineweaver-Burk
1976
EFFECTS OF NON-SUBSTRATE PEPTIDES ON PIG PEPSIN
20
x
I0
10-3 x 1/[S] (M-1)
Fig. 11. Non-competitive activation of release of Gly-Phle
from Gly-Phe-Phe by Tyr-Leu-NH2 (37°C, 30min)
[Pepsin] = 0.6mg/ml; (a) pH3.4; (b) pH2.0;
modifier;
A,
o, no
1 mM-Tyr-Leu-NH2; *, 6mM-Tyr-Leu-NH2.
plots which were used for the calculation of kinetic
parameters shown in Table 4 are given in Fig. 11.
Once again, if linear curves are drawn through the
points at low substrate concentrations it appears that
the modifiers do not change the Km value. However,
as in the case shown above (Fig. 3), modifiers cause a
substrate activation at higher substrate concentrations. The activation constants (Ka) were again calculated as described by Wang et al. (1974).
Discussion
We have presented some initial experiments which
show that a non-substrate peptide can act as activator
of the cleavage of small substrates by penicillopepsin
(Wang et al., 1974). The binding of the modifier
peptide is associated with a marked conformational
change which can be detected by changes in the
circular-dichroism spectrum. The present paper
reports a detailed study of the effect of modifier
peptides on the homologous enzyme, pig pepsin.
The present study shows aboye all the complex
nature of the active site of pepsin and the multiple
effects which binding of modifiers, substrates and
products have on the nature -of the reaction. (The
'active site' is defined as the whole area of the pepsin
molecule where binding of substrate and modifier
peptides occurs, as well as the point where catalysis
Vol. 153
709
takes place. The term 'catalytic site' will be used to
designate those residues that are directly responsible
for the bond-making and -breaking processes.) At
this stage the experiments raise more questions with
regard to the overall mechanism of action of pepsin
than they provide answers. They do, however,
strengthen the proposal that has been made by
Takahashi & Hofmann (1975) who suggested that
the nature of the pathway of the reaction (intermediate formation) is dependent on the nature of the
substrate and that it is highly probable that there is
not one uniform reaction mechanism for pepsin and
for the related enzyme, penicillopepsin. The present
study shows that closely similar peptides can act as
modifiers, can undergo hydrolysis, acyl-transpeptidation, amino-transpeptidation or condensation reactions. Many of these reactions are found to occur in
the same system and thus make the interpretation of
kinetic experiments very difficult.
There are nevertheless a number of points which
emerge clearly from the experiments described. The
most striking is, first, that the modifier peptides
increase acyl- and amino-transfer reactions in all
cases more than hydrolytic reactions. The implication of this on the question of the intermediates will
be discussed below. Secondly, the modifiers are
more effective at higher pH than at low pH. Thirdly,
kinetics of the initial cleavage rates show that the
modifiers have no effect on Km but increase the catalytic rate constants, and, fourthly, there is evidence
in the kinetic experiments that the modifiers can
induce, or at least enhance, a marked substrate activation. A detailed discussion of these points follows.
To take the third point first. That the modifiers
have no effect on Km values agrees very well with the
experiments summarized by Fruton (1970) on the
effect of a secondary binding of long substrates into
extended binding sites. Fruton (1970) showed clearly
that binding in the secondary sites has little effect on
Km. It is reasonable to assume that the modifier
peptides also bind in the same secondary sites as the
peptide chain of long substrates. As Fruton (1970)
suggested, binding in the secondary binding site
probably causes a conformational change that leads
to an increase in the catalytic efficiency of the enzyme.
As has been mentioned above, such a conformational
change on binding of a modifier peptide could be
demonstrated for penicillopepsin (Wang et al., 1974).
We attempted to see whether we could observe a
similar conformational change on binding of some of
the modifier peptides described in the present paper.
In contrast with penicillopepsin, however, where the
circular-dichroism spectrum between 250 and 320nm
offers a sensitive indicator of localized conformational
changes, the circular-dichroism spectrum of pig
pepsin in the near-u.v. range is relatively featureless
and consists largely of one broad positive ellipticity
peak which is complex, as has been shown by
710
Perlmann & Kerwar (1973). Although small changes
were observed when Cbz-Leu-Leu was added to
pepsin, the changes were too small to serve as a useful
probe for the study of interaction of modifier peptides
with pepsin.
Nature of binding site
The studies of Fruton (1970) show clearly that the
secondary binding site extends on both sides of the
catalytic site and that binding on either side leads to
increases in kcat.. The experiments with the modifier
peptides confirm the presence of two separate binding
sites [sites A and B as defined by Takahashi &
Hofmann (1975)]. For instance, Cbz-Leu-Leu and
Cbz-Leu-Met;bind in the secondary site A [in sites
S2 and S3 as defined by Berger & Schechter (1970)]
when they act as modifiers (Figs. 1 and 2, Table 3)
because they activate the cleavage of Leu-Tyr-NH2,
which would bind in sites SI and S,'. More convincing evidence that Cbz-Leu-Met binds in the A
site for activation is contained in Fig. 7 where the
activation of the cleavage of Leu-Trp-Met-Arg by
Cbz-Leu-Met is shown. As has been discussed (Wang
& Hofmann, 1976) the formation of Leu-Leu-Leu
requires that when the precursor Leu-Leu-Leu-TrpMet-Arg is. formed at least four subsites (Si' to S4')
are occupied. For Cbz-Leu-Met to be effective, therefore, it must bind in the secondary binding site A. In
addition Cbz-Leu-Leu inhibits the cleavage of GlyPhe-Phe which presumably occupies sites S2, St and
Si,.
In contrast Tyr-Leu-NH2, Tyr-Tyr-NH2 and
Tyr-Phe-NH2 bind primarily in the binding site B
because they activate the cleavage of Cbz-Phe-Leu.
The decrease in activation at higher concentrations
and eventual inhibition is presumably caused by
competitive binding in site Si', a site which is known
to have a high affinity for aromatic residues (Fruton,
1970). Tyr-Leu-NH2 presumably binds in subsites
S2' and S3'.
The kinetic- experiments (Figs. 3 and 11) also bear
on, the question of multiple binding sites. The
Lineweaver-Burk plots used for determining the
activation constants show deviations from linearity
which are most readily explained by assuming that
the modifiers induce a substrate activation. For
example, in Fig. 3, where the initial rates of release of
Tyr-NH2 from Leu-Tyr-NH2 are measured, the
modifier Leu-Gly-Leu at 2 and 6mM gives Lineweaver-Burk plots that curve downwards markedly
at the higher concentrations. Similarly in Fig. 11,
where the substrate is Gly-Phe-Phe and the initial
rates of release of Gly-Phe are measured, Tyr-LeuNH2 induces substrate activation. In terms of the
binding site this probably means that the modifiers
are bound in one of the secondary binding sites
(Leu-Gly-Leu in site A, or Tyr-Leu-NH2 in site B)
70T.-T. WANG AND T. HOFMANN
and cause a conformational change that not only
leads to an increase in kcat. but also induces binding
of a second molecule of substrate in the other
secondary site (B or A respectively). This in turn leads
to a further enhancement of kcat.. This interpretation
affords another means of estimating the minimum
length of the whole active site. Since Leu-Gly-Leu
probably binds in subsites S4-S2 and Leu-Tyr-NH2
in sites S2' and S3' the minimum length must be seven
amino acids. In the preceding paper the estimated
length based on the nature of transpeptidation
reactions was eight to nine amino acids (Wang &
Hofmann, 1976).
The modifier-induced substrate activation also
simulates the increase of kcat. shown when substrates
of increasing chain length are compared (Fruton,
1970). Increase in the length of substrates on either
side of the scissile bond lead to increased kcat. values.
These increases are additive.
Although no systematic attempt has been made to
obtain information on the side-chain requirements of
modifier peptides, the present experiments do indicate
that hydrophobic residues are the most effective
activators. Thus the tetrapeptide Ala-Ala-Ala-Ala
has little effect on the cleavage of Met-Tyr-Phe-NH2
(Table 2) and no effect at all on the cleavage of
Gly-Phe-Phe (Table 3). Phe-Gly-Gly-Phe also is not
an effective activator. It is also noteworthy that the
amino acid amides Leu-NH2 and Tyr-NH2 have no
effect on Gly-Phe-Phe cleavage, although we have
shown previously that Leu-NH2 and Tyr-NH2 can
act as acceptors or as partners in a condensation
reaction (Wang & Hofmann, 1976) and thus they
presumably bind to the enzyme, but in a way in
which there is no effect on. the activity. Although the
preference of the primary site S.' for aromatic amino
acids has been well documented (Fruton, 1970), the
results of the present study also indicate that subsite
S2' has good affinity for tyrosine. Fig. 10 shows the
effect on the cleavage of Gly-Phe-Phe by the
modifiers Tyr-Leu-NH2, Tyr-Phe-NH2 and Leu-LeuNH2 and it is clear that Leu-Leu-NH2 is the least
effective of the activators. The most effective of three
related modifiers (Tyr-Leu-NH2, Tyr-Phe-NH2 and
Tyr-Tyr-NH2, Fig. 9) is Tyr-Tyr-NH2. This indicates
either a higher affinity of site B for tyrosine or a more
pronounced influence of the tyrosine residues on the
activity.
In contrast leucine- and methionine-containing
modifiers are more effective activators in site A
(cf. Cbz-Leu-Leu in Tables 2 and 3).
The results reported in the present paper also
provide an explanation for the lag phenomena that
were shown both with amino and with acyl transfer
in the preceding paper (Wang & Hofmann, 1976).
The most likely explanation for these lags is that the
transpeptidation products Leu-Leu, Leu-Leu-Leu
and Met-Met and Met-Met-Met act as modifiers for
1976
EFFECIS OF NON-SUBSTRATE PEPTIDES ON PIG PEPSIN
the treaction and cause activation of the nature observed in the experiments described in the present
paper.
pH-dependence of modifier effects
The pH-dependence of the modifier effect shows
unambiguously that all reactions are affected more at
higher pH then at lower pH values. This is shown in
Figs. 4, 5, 8, 9 and 10 and in Table 4. Nevertheless the
cleavage of Gly-Phe-Phe remains optimum at pH3.4
in the presence or absence of modifier (Tyr-LeuNH2).
This is because kc,t. for the cleavage of Gly-PhePhe shows little pH-dependence, an observation that
has been made with a variety of substrates in which
the Phe-Phe bond was cleaved (Hollands et al., 1969;
Sachdev & Fruton, 1969), although in these cases
there was usually some decrease in Km with increasing
pH. More pronounced decreases with increasing pH
were observed by Hunkapiller & Richards (1972)
with trifluoroacetyl amino acids. A particularly
noteworthy point is that Cbz-Leu-Met at pH2
induces the appearance of the transpeptidation product Leu-Leu-Leu from the substrate Leu-Trp-MetArg (Fig. 8). Leu-Leu-Leu is not observed in its
absence; instead, as was shown (Wang & Hofmann,
1976), Leu-Leu is formed as the transpeptidation
product. Also noteworthy is that Cbz-Leu-Met has no
significant influence on the rate of hydrolysis of
leucine from Leu-Trp-Met-Arg or on the formation
of Leu-Leu, either at pH2 or 3.4. The major effect of
the modifier is clearly on the formation of the tripeptide. The experiment shown in Table 6 also highlights the point that pH has little or no effect on Km
and in turn Km is not affected by the presence of the
mnodffier. In the absence of extensive direct information on possible pH-controlled conformational
changes there is no ready interpretation of the pH
effects. Although the modifier effects at any one pH
value are most likely caused by conformational
changes, it is not clear why the effects are more
pronounced at the higher pH values. The only
relevant conformational information is the study of
the pH-dependence of the circular-dichroism spectrum by Perlmann & Kerwar (1973). These workers
showed a marked difference in the spectrum at low
wavelength between pH 1.2 anid 4.6. The presence of
N-acetylphenylalanyl-3,5-di-iodotyrosine had no influence on the pH4.6 spectrum, but markedly
altered the spectrum at pH 1.2. It is possible that the
dipeptide binds as modifier as well as a substrate and
therefore it is not possible to ascribe the observed
circular-dichroism change definitely to binding in
either the primary or one of the secondary sites.
Implications for the mechanism
The central question in all studies relating to the
action of pepsin is 'What, if any, covalent interVol. 153
711
mediates are involved in any particular reaction?'
This question has become especially prominent since
evidence has come forward for transpeptidation
reactions that proceed in very high yield via acyl
intermediates (Wang & Hofmann, 1976). At present
the evidence that covalent acyl or amino intermediates
respectively are involved in acyl or amino transpeptidations is reasonably good. However, for hydrolytic
reactions, especially of good substrates, there is as
yet no evidence as to which, if any, covalent intermediates are on the pathway (Silver & Stoddard,
1975). Hofmann (1974) and Takahashi & Hofmann
(1975) proposed that the nature of the substrate has a
strong influence on the nature of the pathway of the
reaction, which can proceed via one intermediate or
another or possibly via no intermediates at all. The
experiments described in the present paper have a
bearing on this question to the extent that the modifier peptides simulate to some degree the extended
substrates studied by Fruton (1970), which depend on
secondary binding for high catalytic efficiencies. Let
us assume that the activating effect of the modifier
peptides simulates the effect of the secondary binding
of long substrates and let us further assume that the
degree of the transpeptidation reactions observed is
an indicator of the stability of a covalent intermediate.
In this case the results of the present study strengthen
the case for a mechanism that involves covalent
intermediates, because the modifiers exert their overall
activating influence predominantly through an
activation of the transpeptidation reactions. The
increased stability of the intermediates is probably
due to the fact that the modifiers, which are mostly
composed of hydrophobic amino acids, cause
conformational changes that increase the hydrophobic nature of the active site, and that could go so
far as virtually to exclude water from the catalytic
site. This could especially explain the stability of the
acyl intermediate which is a putative anhydride. This
expulsion of water could explain the very high yield
of transpeptidation products as compared with
hydrolysis.
The major uncertainty about the involvement of
covalent intermediates in the hydrolysis of good
substrates is that the transpeptidation reactions of
both acyl- and amino-transfer types have been
observed mostly with substrates in which the two
N-terminal or C-terminal amino acids respectiv'ely
are amino acids with long hydrophobic side chains,
except for Cbz-Glu-Tyr (Neumann et al., 1959).
Further, transpeptidation reactions involve the
transfer of a single terminal amino acid only, except
possibly for Ac-Phe-Phe-Gly (Kitson & Knowles,
1971), who studied the reaction Ac-Phe-Phe-Gly+
Ac-[3H]Phe a± AcPhe+Ac-_3H]Phe-Phe-Gly. In this
case, however, the possibility of transfer by a
substrate-product equilibrium via condensation has
not been excluded, and there is no evidence for a
712
covalent intermediate. It can be argued that the
presence of a free terminal ammonium or carboxylate
group induces a state in the catalytic site that makes
the formation of the corresponding covalent intermediate possible, whereas in the absence of a terminal
ammonium or carboxyl group the catalytic site is in
a state in which covalent intermediates are not formed.
The effect of the modifiers would then be to increase
transpeptidation by favouring the induction of the
appropriate catalytic state. Some initial attempts have
been made to find a modifier that could activate the
cleavage of substrates for which no transpeptidation
has been observed. The effects of Leu-Leu-Leu on
Gly-Gly-Phe-Phe-Gly, and of Tyr-Leu-NH2 on
Cbz-His-Phe-Trp-OEt were studied. In both cases
low modifier concentrations (about 1 mM) had no
significant effect (less than 10 % activation) and higher
concentrations inhibited the reaction. There are
two probable major reasons for the failure of these
experiments. The first is that the modifiers can no
longer bind in the secondary sites because the terminal
residues of the substrate occupy positions S2 and/or
S2' and thus prevent binding of the modifier. The
second is that the modifiers can only activate the
formation of acyl or amino intermediates respectively
from terminal amino acid residues. The latter possibility is, however, in direct contradiction to the
postulate that the modifiers simulate the secondary
binding effects of long substrate where overall
cleavage is accelerated.
Further work is clearly needed before these alternatives can be evaluated productively.
It is noteworthy to point out also that activation
phenomena have been observed when the acid
proteinase of Endothia parasitica acts on small
substrates (i.e. Leu-Leu-NH2; Whitaker & Caldwell,
1973) as judged from kinetic experiments. Presumably this is due to effects in the secondary binding
sites. Other enzymes in which effects of secondary
binding on catalytic rate constants have been observed
are chymosin, Rhizopus pepsin (Voynick & Fruton,
1971) and cathepsin D (Ferguson et al., 1973). It
appears therefore, that acid proteinases in general
have as a common feature secondary binding sites
through which modulation of the catalytic properties
occurs.
This work was supported by the Medical Research
Council of Canada (Grant MT-1982). T-T. W. was the
holder of an M.R.C. (Canada) Fellowship. We are
grateful to Mr. C. Yu for his assistance with the amino
acid analyser.
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1976