277s
Clinicalscience (1981) 61,277s-280s
Complex competitive and non-competitive inhibition of rat lung
angiotensin-converting enzyme by inhibitors containing thiol
groups: captopril and SA 446
F. A. 0. M E N D E L S O H N , J. C S I C S M A N N A N D J . S . H U T C H I N S O N
University Department of Medicine, Austin Hospital, Heidelberg, Victoria,Australia
Summary
1. The kinetics of the inhibitory action of four
different angiotensin-converting enzyme inhibitors was evaluated in vitro with rat lung enzyme
and the substrate hippuryl-histidyl-leucine.
2. Enzyme velocity against substrate concentration curves were fitted directly to hyperbolae by a weighted least squares iterative
method to obtain apparent values of K,, V,,.
and K/V at each concentration of inhibitor.
3. The inhibitory constants K, and Ki were
obtained by weighted h e a r regressions of K/V
against i and 1/V against i respectively.
4. Teprotide was the least potent inhibitor with a
K , of near 20 nmol/l whereas captopril (SQ
14 225) and SA 446 were both approximately 10
times and MK 421 approximately 20 times more
potent.
5. Two inhibitors which lacked thiol groups
[teprotide or SQ 20 881 and N-(1-S-1-carboxy3-phenylpropyl)-~-Ala-~-Pro
or MK 4211 produced a purely competitive pattern of inhibition
with increased apparent K , but no change in
apparent V,,.
6. Two inhibitors containing thiol groups
[captopril or SQ 14 225 and 2-(2'-hydroxypheny1)- 3 - (3 -mercaptopropanoyl)-4-thiazolidine
carboxylic acid or SA 4461 both produced a
mixed competitive and non-competitive pattern of
inhibition with increased apparent K , and decreased Vmax..
7. It is possible that thiol-containing inhibitors
might produce non-competitive inhibition of
converting enzyme by forming strong bonds with
zinc near the active site of the enzyme.
Correspondence: Dr F. A. 0. Mendelsohn, University
Department of Medicine, Austin Hospital, Heidelberg,
Victoria 3084, Australia.
Key words: angiotensin-converting enzyme,
captopril, enzyme inhibitors, enzyme kinetics,
MK 42 1, SA 446, teprotide.
Abbreviations:
enzyme.
ACE,
angiotensin-converting
Introduction
The development of a series of angiotensinconverting enzyme (EC 3.4.15.1; ACE)
inhibitors [ 11 has excited much interest. The most
potent of these inhibitors contains a thiol group
[l], which was postulated to complex with the
zinc atom near the active site of the enzyme [21.
The thiol-containing inhibitors (captopril or SQ
14 225; i2-41) and mercaptoacyl derivatives of
thiazolidine carboxylic acid (YS 980 [51) or SA
291 [6] have been reported to be purely competitive inhibitors of ACE. In view of the fact that
thiol-containing compounds may form exceptionally strong bonds with zinc [71, the reported
purely competitive nature of these inhibitors is
surprising. However, the published kinetic data
on these compounds is rather limited and often
the conclusions seem unjustified in view of the
methods used for analysis of the data. We
therefore decided to reinvestigate the kinetics of
inhibition of ACE by two inhibitors containing
thiol groups: captopril or SQ 14 225 and SA 446
and two inhibitors which did not contain thiol
groups: teprotide or SQ 20 881 and MK 421 [Sl.
Methods
Inhibitors and reagents
Teprotide (SQ 20 881, batch no. NN 012ND)
and captopril (SQ 14 225, batch no. NN 01 1NB)
were generous gifts of the Squibb Institute
278s
F. A . 0.Mendelsohn, J . Csicsmann and J. S . Hutchinson
KApp./Vapp.against i and l/VaPP. against i
(Princeton, NJ, U.S.A.). SA 446 [2-(2'-hydroxyrespectively I13I.
pheny1)-3 - (3 -mercaptopropanoyl)-4-thiazolidine
carboxylic acid] was a gift from the Santen
Pharmaceutical Co. (Osaka, Japan). MK 421
Results
[N-(1-S- 1-carboxy-3-phenylpropyl)-~-Ala-~-Prol
As shown in Table 1, SQ 20 881 produced a rise
[81 was a gift from the Merck Institute (Rahway,
in KAPP. (P< 0.001) with no change in Vapp.
NJ, U.S.A.). Hippuryl-histidyl-leucine (Hip-His(P > 0.05), indicating that it acted as a purely
Leu) was from Vega Fox (Tucson, AZ, U.S.A.)
competitive inhibitor. In contrast, captopril proand o-phthaldialdehyde from Sigma.
duced a rise in K%P. (P < 0.001) but also a fall
Rat lung was homogenized in phosphate buffer
and a microsomal fraction prepared [91. The
in V a p p . (P< 0.02) and a rise in K2p./VapP.
specific activity of the crude preparation was 50
( P < 0.02); this pattern corresponds to a mixed
competitive and non-competitive inhibitor [ 131.
nmol of substrate hydrolysed min-' mg-' of
SA 446 produced a similar mixed pattern of
protein. The preparation was diluted 10-fold in
inhibition with a rise in KZP. (P< 0-025) and rise
sodium phosphate buffer before use.
in KAPP./VaPP. ( P < 0.01). MK 421 produced a
purely competitive pattern, as shown by a rise in
Kinetic experiments
Kipp. (P < 0.005) and K;Pp'/Vapp. (P < 0.001)
with no change in Vapp.(P> 0.1).
Fixed concentrations of the diluted lung
enzyme (10 pl containing 29 pg of protein) were
K , for S Q 20 881 was near 20 nmol/l whereas
incubated with 250 p1 of Hip-His-Leu (5.0, 2-0,
that for captopril and SA 446 were approxi1.O or 0.5 mmol/l) in potassium phosphate buffer
mately 10-fold less. MK 421 appeared the most
(100 mmol/l), pH 8.3, containing NaCl (300
potent inhibitor with K , near 1 nmol/l. K; was
mmol/l) for 15 min at 37OC. The histidyl-leucine
near 5 nmol/l for both SQ 14 225 and SA 446
(His-Leu) liberated was assayed by forming a
(Table 1).
fluorescent adduct with o-phthaldialdehyde [ 101,
which was measured at excitation and emission
Discussion
wavelengths of 360 and 500 nm with a Turner
model 430 fluorimeter against a standard curve
The experiments reveal that the two inhibitors
of His-Leu (0-10 nmol). Generation of His-Leu
which contain thiol groups, captopril and SA
was linear with time over this period. Recovery of
446, both produced a mixed pattern of comHis-Leu (5 nmol) added to the assay mixture was
petitive and non-competitive inhibition. By con96.4% (SD 10.1, n = 6), indicating stability of the
trast, the two inhibitors devoid of thiol groups,
cleavage product. Between-assay coefficient of
SQ 20 881 and MK 421, produced purely comvariation was 10.9% ( n = 18).
petitive inhibition.
Substrate velocity curves were obtained withSQ 20 881 has been reported to be a purely
out inhibitor (control) and in the presence of two
competitive inhibitor of rabbit lung converting
different concentrations of inhibitor in each
enzyme with a K,of 0.1 pmol/l [14]. Captopril
experiment.
h w been reported to act as a competitive
inhibitor of the rabbit lung enzyme with a K ,
identical with that found in the current experiStatistical analysis
ments [l]. Kinetic studies of SA 446 have not
The enzyme velocities at the different substrate
been published but other mercaptoacyl
concentrations were fitted directly to an hyperderivatives of thiazolidine carboxylic acid
bola by a least .squares iterative procedure with a
(YS 980 151 or SA 291) have been reported [6] to
program written in Fortran [ l l , 121. This
be competitive inhibitors. The K , value for
provided values of apparent K , (K:'.), Vmax.
MK421 reported here agrees closely with the
IC,, value reported by Patchett et al. [81.
( VaPp.)and K / V (KZP./Vapp.) at each inhibitor
Most of the published studies of these comconcentration (i). The means and SEM of these
pounds have apparently used visual inspection of
parameters were calculated from the results of
Lineweaver-Burk plots to determine the type of
separate experiments and the significance of
changes from control was evaluated by Student's
inhibition. This procedure is prone to large errors
in estimating the Michaelis-Menten parameters
unpaired t-test.
12, 151 and might easily fail to detect the change
The type of inhibition was assessed from the
in apparent V,,,. observed with thiol-containing
changes in these apparent kinetic parameters
inhibitors in the current study.
[131. K , and K,' were calculated for each
A number of thiol compounds have been
experiment from a weighted Linear regression of
Converting enzyme inhibitors
279s
TABLE1. Kinetic properties off o u r converting enzyme inhibitors
Mean results k SEM are shown with the number of experiments in parentheses.
Inhibitor
SQ 20 881
Inhibitor
concn.
(nmolll)
Apparent K ,
(mmol/l)
Apparent ,f‘
(nmol min-’ mg-I
of protein)
IO’K/V
Type of inhibition
and inhibition constants
(nmolll)
0
1.78 k 0.22
(3)
5.34 C 0.28
(3)
8.43 f 0.23
(3)
1.99 f 0.08
51.5 f 2.1
(3)
44.1 f 1.8
(3)
42.0 f 2.8
(3)
52.5 k 2.8
(4)
36.4 k 4.6
(3)
30.1 f 5.6
(4)
44.1 f 5.3
(8)
36.4 f 3.9
(8)
23.5 f 3.5
3.46 f 0.49
(3)
12.2 f 1.0
(3)
20.3 1.7
(3)
3.80 f 0.11
(4)
6.63 f 0.57
Purely competitive
(4)
K;= 5.06 f 0.98
50
100
SQ 14 225
0
(4)
1
5
SA 446
0
1
2.43 k 0.47
(3)
4.63 f 0.34
(4)
2.06 k 0.36
(8)
2.90 f 0.41
(8)
5
MK 421
(parent diacid)
0
I
5
4.30f 0.81
(8)
2.74 f 0.31
(6)
6.31 f 1.34
(6)
26.2 f 6.4
(5)
(8)
45.8 f 4.1
(6)
38.9 f 4.1
(6)
69.3 f 13.9
(6)
reported to inhibit angiotensin-converting enzyme
[ 16-19], presumably by interacting with zinc
near the active site in the enzyme [20, 211.
Thiol-containing compounds can form very
strong bonds with zinc 171; this interaction has
been postulated to explain the high potency of
captopril [2]. We believe it is therefore not
unexpected that some of the high affinity thiol
inhibitors of converting enzyme would show
some non-competitive effects.
The biological significance of the type of
inhibition of converting enzyme produced by
these compounds is not known, but it is of
interest that the plasma concentrations of captopril observed in man after therapeutic use of the
drug [22] are more than 5000 times greater than
those which showed non-competitive inhibition in
vitro in these experiments.
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12.3 k 3.2
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