MECHANISM OF INHIBITION. OR PEPSIN BY PEPSTATIN
MECHANISM OF INHIBITION. OR PEPSIN BY PEPSTATIN
Although the binding is not of the covalent nature, the inhibition roughly
follows the stoicheometrical mode. Pepstatin can be used to titrate pepsin.
Formation of an equimolar pepsin-pepstatin complex can be shown by gel
filtration. Diacetylpepstatin, which has weaker activity than pepstatin com-
petitively inhibits pepsin with a dissociation constant of 7.3XlO"6M. Data of
pepstatin binding of chemically modified pepsins suggested that pepstatin binds
with the active site surrounded by two aspartic acid moieties.
Pepstatin, isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-
alanyl-4-amino-3-hydroxy-6-methylheptanoic acid, was discovered in streptomyces
culture nitrates and has been shown to be a specific inhibitor of acid proteases1*2'^.
It shows a strong protective effect against pylorus-ligated rat stomach ulcer, and an
effect against human gastric ulcer has been observed in clinical studies. A specific
inhibitor of acid proteases is a biochemical tool useful for analysis of the role of
these enzymes in stomach and duodenal ulcers. In this connection, the mode of
inhibition of pepsin is thought to be interesting. In this paper, we report the strong
binding of pepstatin with pepsin in the equimolar ratio and our studies suggesting
the site in pepsin binding with this inhibitor.
Materials and Methods
Enzyme, inhibitors and others
Pepsin (twice crystallized) was purchased from Sigma Chemical Co., U.S.A. (lot 28B-
1900) and used without further purification. Pepstatin was prepared by fermentation of
Streptomyces argenteolus var. toyokaensis as previously described1). Diacetylpepstatin was
prepared from pepstatin as previously described1). Radioactive pepstatin (specific activity
was 2.2X105dpm//zg) was kindly prepared by Dr. T. Komai, National Institute of Health,
Tokyo, by exposing pepstatin to tritium gas. N-Acetyl-L-phenylalanyl-L-diiodotyrosine
(APDT) was purchased from Sigma Chemical Co., U.S.A., Sephadex G-50 (medium) from
Pharmacia, Sweden, and />-bromophenacyl bromide from Tokyo Kasei Kogyo Co., Japan.
All other reagents employed were analytical grade.
Determination of the initial velocity of peptic hydrolysis
The Anson method slightly modified as previously described2) was used for the assay
of peptic activity, using hemoglobin as the substrate. For enzyme titration and kinetic
study APDTwas used as the substrate, because it was the most rapidly hydrolyzed of the
peptide substrates commercially available. A 2mMAPDTsolution in 4mMNaOHwas
diluted before used to the desired concentration with 0.01N HC1. To each test tube 3.2 ml ot substrate solution containing desired amount of pepstatin or diacetylpepstatin was
added and preincubated for 3minutes at 37°C. The reaction was started by addition of
0.2ml of 0.001N HC1 containing 60jug of pepsin except as otherwise noted. After the
incubation at 37°C peptic hydrolysis was stopped by addition of 0.1ml of 0.7N NaOHand
the released diiodotyrosine was determined by ninhydrin method slightly modified by
Jackson et al^. The substrate concentration used was 10"4M for enzyme titration experi-
ment but in case of the kinetic study it was varied in a range of 5~16xlO~5M.
Binding of 3H-pepstatin to pepsin
The mixture of pepstatin and pepsin was passed through a column of Sephadex G-50
(2X55cm) equilibrated with eluting buffer at a cold room. Three kinds of eluting buffers
were employed. They were 0.05m KC1-HC1buffer, pH 2.0, 0.05m acetate buffer, pH 5.5,
and 0.05m Tris-HCl buffer, pH 7.2. The eluate was fractionated every 3g by a fraction
collector. Protein content of each fraction was determined by absorption at 280mju and
pepsin activity of each fraction was measured by hydrolysis of hemoglobin. The radioac-
tivity of 3H-pepstatin in 0.1ml of each fraction was determined in a Beckman Liquid
Scintillation System using 6ml of Bray's scintillation solution5). The inhibitory activity
of pepstatin was determined by the method previously described2).
Preparation of chemically modified pepsins
Two aspartic acid residues in pepsin active site were modified by esterification with
^>-bromophenacyl bromide or/and <2-diazo-/>-bromoacetophenone as described by Erlanger
et al.G) Modification was confirmed by the determination of hemoglobin hydrolyzing
activity.
Results and Discussion
The inhibitory activity of pepstatin is dependent on the concentration of pepsin
as described in a previous paper2). When pepsin was used at a concentration of 2,
0.5, 100, 18//g/ml for hydrolysis of casein, hemoglobin, N-acetyl-L-phenylalanyl-L-
tyrosine or N-acetyl-L-phenylalanyl-L-diiodotyrosine, respectively, pepstatin concen-
tration exhibiting 50 % inhibition was 1.5xlO"8M, 4.5X10"9M, l.lxlO"6M or 2.3xlO"7M,
respectively. This proportionality of pepstatin concentration to pepsin concentration
suggests that pepstatin would bind to pepsin tightly. The evidence supporting this
suggestion will be described in this paper.
The rates of peptic hydrolysis of APDTat different pepsin
concentrations with a constant amount of
pepstatin were determined and plotted against
the amount of pepsin. Two straight lines in
the presence of pepstatin have identical slope
with a straight line determined in the absence
of pepstatin. This plot suggests irreversible
binding, but pepstatin is a reversible inhibitor
because dialysis for 2days of pepsin-pepstatin
complex separated by Sephadex gel nitration
releases free pepstatin out of the dialysis bag,
and pepstatin is recovered from the complex
by extraction with butanol or by standing in
alkaline condition inactivating pepsin. It is
thought that pepstatin binds to pepsin so
tightly that the dissociation
of enzyme-inhibitor complex
is very slow. The amounts
of pepsin indicated at the
intersection with the hori-
zontal line in the presence
of pepstatin in Fig. 1 must
be equivalent to the amount
of inhibitor added. As a
method of titrating pepsin
normality has never been
established, pepstatin is very
useful for this purpose. In
this experiment 1jug of
pepstatin was equivalent to
65 jug of pepsin. Considering
that the molecular weights
of pepstatin and pepsin are 686 and 35,000, respectively, it is concluded that one
molecule of pepstatin combines with one molecule of pepsin and the active pepsin
molarity used in this experiment was calculated to be 77% of that calculated from
the weight.
It was also proven by another experiment with Sephadex G-50 gel filtration that
pepstatin is a tight binding inhibitor. Pepstatin labeled with tritium was used and
the experimental condition in detail is described in the legend to Fig. 2. The mixture
of pepsin and 3H-pepstatin were incubated at 37°C for 10minutes and passed through
Sephadex G-50 column. As shown in Fig. 2c) 3H-pepstatin appeared in two peaks.
The second peak corresponded to free pepstatin (Fig. 2a) (and the first peak corre-
sponded to that of pepsin (Fig. 2b)). It means that 3H-pepstatin binds to pepsin
tightly. Pepsin activity in this pepsin-pepstatin complex was 1.3% of free pepsin
activity in hemoglobin hydrolizing activity. The same result was obtained at pH 2.0
and pH 5.5. When 0.05m Tris-HCl buffer of pH 7.2 in which pepsin was denatured
was used as eluting buffer, pepstatin did not bind to pepsin. Pepstatin did not bind
to pepsinogen, so it was concluded that pepstatin binds tightly to active form of
pepsin.
Since inhibition of pepsin by pepstatin appears to be of the pseudo-irreversible
type, kinetic analysis has many limitations. Noncompetitive inhibition in Lineweaver-
Burk plot and upwards curvature in Dixon plot reported in our preliminary paperX)
can be explained as mutual depletion system7). Fromthe results described in this paper
our previous conjecture of multiple binding of pepstatin to pepsin should be corrected.
Diacetylpepstatin is less active than pepstatin and can be used for kinetic studies.
Diacetylpepstatin was a competitive inhititor as shown in Lineweaver-Burk plot and
Dixon plot (Fig. 3) using APDTas the substrate. Its dissociation constant was calculated.
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