From the
In the zinc metallopeptidases produced by the genus Bacillus, an active site histidine has been proposed to
either stabilize the transition state in catalysis by donating a
hydrogen bond to the hydrated peptide (Matthews, B. W.(1988) Acc. Chem. Res. 21, 333
-340) or to polarize a water
molecule, which subsequently attacks the peptidyl bond (Mock, W. L.,
and Aksamawati, M.(1994) Biochem. J. 302, 57
-68).
Site-directed mutagenesis techniques have been used to change this
residue in the zinc endopeptidase from Bacillus
stearothermophillus to either phenylalanine or alanine. At pH 7.0,
the k
The closely related extracellular zinc endopeptidases (EC
3.4.24.27) produced by members of the bacterial genus Bacillus are thermostable, calcium-binding neutral proteases. Two of these
enzymes, from Bacillus thermoproteolyticus (thermolysin) and Bacillus cereus, have been crystallized to a high resolution
and shown to have virtually identical three-dimensional structures and
active site geometries(1) . From molecular modelling studies,
similar structures for other enzymes of the same group have been
predicted, such as those secreted by Bacillus
stearothermophillus(2) , Bacillus
subtilis(3, 4) , and B. subtilis var. amylosacchariticus(5) . An active site model of the
neutral proteases, derived from x-ray crystallographic studies of the
binding of a variety of different inhibitors to thermolysin (for
review, see Ref. 6), has found a wide application, as many mammalian
zinc metallopeptidases, such as neutral endopeptidase 24.11 (EC
3.4.24.11) and angiotensin-converting enzyme (EC 3.4.15.1), appear to
have thermolysin-like active sites. The model has therefore formed the
basis for the design of inhibitors of these physiologically important
enzymes (for review, see Ref. 7).
The bacterial enzymes are bilobal,
and the active site, situated in a cleft between the two lobes,
contains an atom of zinc bound with a tetracoordinate geometry to two
histidines and a glutamate, with a water molecule providing the fourth
ligand. The two histidines are found in a consensus sequence,
HEXXH, present in the majority of zinc endo- and
aminopeptidases(8) . A large hydrophobic pocket in the P`
The mutated codon is
underlined. Mutated pMaMlu were identified by DNA sequencing, and
mutated Mlu 1 fragments were ligated into the plasmid pGB501 Mlu; the
ligation mixture being used to directly transform Bacillus protoplasts. Subsequently, halo-producing clones were identified
and further analyzed by restriction digest analysis and DNA sequencing.
Wild-type and mutant NPrs were purified in a single step
using the Gly-D-Phe affinity column with yields of around 5
mg/liter for the wild-type enzyme and 0.5 mg/liter for H231F-NPr and
H231A-NPr. SDS-PAGE showed, for the three enzymes, the presence of a
single protein with an apparent molecular mass of 36 kDa (Fig. 2). Recovery in each case was estimated to be around 50%,
and the reduced yields of the mutant enzymes are therefore likely to be
due to a decrease in the extracellular levels of the enzymes. The Bacillus neutral proteases are synthesized as preproenzymes,
with the prosequence (200 residues for NPr) probably being removed at
the cell membrane(23) . This is thought to be an autocatalytic
process, and mutations that change enzyme activity might therefore be
expected to affect the levels of mature enzyme produced. This appeared
to be the case for the mutations H231F and H231A, as Western blot
analysis of the culture supernatants, using the monoclonal antibody Mab
T1, showed that the wild-type enzyme was present in higher levels (Fig. 3). In contrast, in total cell extracts, a 60-kDa protein
was detected, only in B. subtilis transformed with the mutated
enzymes, which could correspond to the unprocessed proenzyme. Similar
results were obtained when the active site histidine of the neutral
protease from B. subtilis was mutated(24) .
At pH 7.0 in HEPES buffer, the K
The optimum pH for the degradation of 50 nM
[
The inhibition of [
Structural studies have indicated that His-231 of the Bacillus neutral proteases stabilizes the transition state by
forming a hydrogen bond with the oxygen of the amide bond to be cleaved (Fig. 1A)(6) . On the other hand, a more crucial
function for the residue has recently been proposed from mechanistic
studies, in which it would polarize the attacking water molecule in the
first step of catalysis (Fig. 1B)(9) , a role
previously subscribed to Glu-143(6) . The results presented here
would seem to be in favor of the less critical role for His-231 in
catalysis, as its replacement by either Phe or Ala led to the
production of enzymes that still retained proteolytic activity, albeit
reduced. In addition, this reduction, mostly due to a decreased k
Further evidence for a nonessential role of
His-231 has come from x-ray crystallographic studies of representative
members of the metzincin family of zinc endopeptidases (39) such
as astacin(40, 41) , matrix
metalloproteases(42, 43, 44) , and adamalysin
II(45) , in which no equivalent residue has been found. These
enzymes differ from the thermolysin family in that the three
zinc-binding ligands are histidines contained in an extended consensus
sequence, HEXXHXXGXXH. Despite this
difference, a close topology has been found with thermolysin in the
zinc-binding environment, and it is likely that the two families have
similar mechanisms of action(41, 44) . It has been
proposed that the zinc in the metzincins would have a higher net
positive charge than in thermolysin, where one ligand is a negatively
charged carboxylate, allowing it to play a larger role in stabilizing
the negative charge of the transition state and obviating the need for
an equivalent to His-231(43, 44) . In addition, as with
the thermolysin-like enzymes, mutation of the consensus sequence
glutamate in one of the metzincins, endopeptidase 24.18, led to a total
loss of detectable enzyme activity (46).
The results of the
inhibition studies are also in agreement with x-ray crystallographic
studies of thermolysin. In the thiol-containing inhibitors crystallized
with the enzyme, the distance between the sulfur atom and His-231 as
well as Tyr-157 was found to be too far for hydrogen
bonding(6, 30) , and the absence of such an interaction
with His-231 has been confirmed by the lack of change in the K
Again, for the binding of phosphoramidon, an interaction with
His-231 would be expected from crystallographic
studies(6, 35) . However, for this inhibitor, the
presence of the residue seems to be even more critical. It is unlikely
that inhibitor binding was prevented by any direct compression around
the zinc due to the mutation, as there was a total loss of measurable
binding affinity for both the Phe and Ala mutants. Several hypotheses
can be proposed to account for the loss of affinity observed. The
formation of a hydrogen bond with His-231, and possibly Tyr-157, might
be required to limit the rotation of the P-N-C bonds,
allowing optimal interaction between the oxygen atom of the
phosphoramide group and the zinc cation. Alternatively, the possible
loss of a structured water molecule(s) present in the wild-type enzyme
could be unfavorable for binding to the mutant enzymes, or, conversely,
the presence of additional unstructured water molecule(s) in the active
sites of the mutated enzymes could have a similar effect. Further
studies, including those of molecular modelling, will be required to
ascertain the exact role of His-231 in phosphoramidon binding.
Reactions were
carried out in 50 mM HEPES buffer pH 7.0 as described under
``Experimental Procedures.''
We thank Dr. C. Vita (CEA-Saclay-France) for providing
some of the thermolysin peptides used in antibody mapping and C. Dupuis
for invaluable help in preparing this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/K
values of
the substrate leucine enkephalin for the phenylalanine and alanine
mutants were reduced by factors of 430- and 500-fold, respectively, as
compared with the wild-type enzyme, mostly due to changes in k
. In addition, the enzymatic activities of the
mutant enzymes showed little pH dependence in the alkaline range,
unlike the wild-type enzyme. The mutations did not greatly alter the
binding affinities of inhibitors containing sulfydryl groups to chelate
the active site zinc, while those of inhibitors containing hydroxamate
or carboxylate zinc-chelating groups were increased between 80- and
250-fold. The largest change in the binding affinity of an inhibitor
(>5 orders of magnitude) was found with the proposed transition
state mimic, phosphoramidon. The results are generally in agreement
with x-ray crystallography studies and favor the involvement of the
active site histidine in transition state binding.
position confers a preference for cleaving peptides at the
N-terminal side of hydrophobic residues, and from the x-ray data it has
been deduced that catalysis proceeds by a general base-type mechanism,
with the zinc-bound water molecule being displaced toward the glutamate
of the consensus sequence (Glu-143)
(
)by the
incoming substrate. The resulting polarization of this water molecule
then leads to an attack of the carbonyl carbon of the scissile bond,
with the negative charge that develops on the carbonyl group being
stabilized by hydrogen bonds formed with both His-231 and Tyr-157 (Fig. 1A)(6) . However, an alternative catalytic
mechanism has recently been proposed, based on kinetic studies, in
which His-231 acts initially as the general base that polarizes the
attacking water molecule, with Glu-143 possibly playing a role in
charge neutralization (Fig. 1B)(9) .
Figure 1:
Proposed roles of His-231 in NPr
catalysis. A, in stabilizing the transition state (adapted
from Matthews (6)); B, in polarizing a water molecule that
would attack the carbon of the peptide bond leading to the formation of
the peptide bond (adapted from Mock and Aksamawati (9)). Presumed
hydrogen bonds are shown as brokenlines, and zinc
interactions as striatedlines.
The
neutral protease produced by B. stearothermophillus (NPr)(
)has an 85% sequence homology with
thermolysin, which includes all of the important active site residues
and, as discussed above, has been predicted to have a similar structure
and active site geometry. In this study, site-directed mutagenesis has
been used to change the active site histidine (His-231) in this enzyme
to either Phe or Ala. Wild-type and mutant enzymes were purified, and
their kinetic and inhibitor binding properties were analyzed.
Materials
All culture media components were
Difco products obtained from OSI (Maurepas, France). Oligonucleotides
were purchased from the laboratory of Professor J. Igolen (Institut
Pasteur, Paris, France). The Sequenase enzyme (version 2) (U. S.
Biochemical Corp.) and [
-
P]dATP were
purchased from Amersham Corp.
[
H]Tyrosyl-glycyl-glycyl-phenylalanyl-leucine
([
H]leucine enkephalin) was from DuPont NEN.
Leucine enkephalin (tyrosyl-glycyl-glycyl-phenylalanyl-leucine) and
phenylalanyl-alanine were purchased from Bachem (Bubendorf,
Switzerland), and phosphoramidon and thermolysin were obtained from
Sigma. The inhibitors, thiorphan(10) ,
retrothiorphan(11) , HACBOGly (12), and ST 51(13) , were
synthesized in the laboratory as described previously. The synthesis of
the other inhibitors, JCH 24 and ES 92 will be published elsewhere. The
structures of these inhibitors are given (see ). Poropak Q
was from Waters (Saint-Quentin Fallavier, France). Other chemicals were
purchased from Sigma or Prolabo (Paris, France).
Bacterial Strains, Plasmids, Bacteriophage, and Culture
Media
The Bacillus strain used throughout was B.
subtilis DB117, lacking neutral protease activity (Emhis nprR2 nprE18 spr A3,
npr
)(3) . For site-directed
mutagenesis experiments, the Escherichia coli strains, WK-6 muts and WK-6, were employed. Npr was produced using the
plasmid, pGE501(Cm
), which contains the entire coding
sequence of the neutral protease(3) . In order to mutate the npr gene, the plasmid pMa (14) containing the Mlu 1
fragment of pGE501 (pMaMlu) was employed, and to reconstitute the npr gene, the mutated fragment was ligated into the unique Mlu
1 site of pGE501 Mlu, which is a derivative of pGE501 in which the Mlu
1 fragment has been deleted. B. subtilis DB117 was propagated
at 37 °C in LB broth or on tryptose blood agar base plates,
containing 5 µg/ml erythromycin. B. subtilis DB117
containing pGE501, or one of its derivatives, was grown in the same
media containing 5 µg/ml chloramphenicol. In order to screen for
protease activity by colony halo formation on Petri dishes, skimmed
milk (1% (w/v)) was included in these media, solidified with 1.5% (w/v)
agar. B. subtilis protoplasts were regenerated on DM3
plates(15) . E. coli were grown routinely on LB broth
at 37 °C with antibiotics, ampicillin, or chloramphenicol being
added to final concentrations of 100 and 25 mg/ml, respectively, when
required.
Plasmid DNA Isolation and Manipulation
Plasmid DNA
was isolated from B. subtilis by a modified form of
the alkaline lysis method described by Bron(16) . Further
purification of large scale plasmid preparations was achieved using a
modified polyethylene glycol precipitation method(17) .
Extraction and purification of double-stranded pMaMlu DNA was performed
using a Quiagen kit. For the production of single-stranded pMa
plasmids, the helper bacteriophage M13K07 was used, and single-stranded
DNA was subsequently extracted using standard methods. DNA sequencing
of double-stranded plasmid DNA was performed using the
dideoxynucleotide termination method (18) using the
Sequenase enzyme (version 2),
[
-
P]dATP, and oligonucleotides
complementary either to parts of the pMa/c vector or to the NprSte
gene.
Transformation of B. subtilis DB117
For plasmid
transformation, B. subtilis protoplasts were prepared as
described by Bron(15) .
Site-directed Mutagenesis
Mutagenesis was
performed using the gapped duplex method (14) in which mutations
are accompanied by a switch in antibiotic resistance. The following
oligonucleotides were employed to mutate His-231 to either Ala or Phe:
H231A, 5` GCT GTT TGT AGC GAC GCC GCC 3`; and H234F, 5` GCT
GTT TGT AAA GAC GCC GCC 3`.
Expression and Purification of the Extracellular
Proteases
B. subtilis DB117 cells harboring
the plasmid pGE501 were grown in LB broth containing 5 mM CaCl and 5 µg/ml chloramphenicol, at 37 °C,
with shaking (180 rpm) for 16 h. Wild-type and mutated enzymes were
purified from the culture supernatant by affinity chromatography using
a column (1
4 cm) of Gly-D-Phe, coupled to
CNBr-activated Sepharose 4B resin following the manufacturer's
instructions and equilibrated in 20 mM sodium acetate, pH 5.5,
containing 5 mM CaCl
. The culture supernatant,
obtained by centrifugation of the culture at 6000
g for 10 min, was slowly adjusted to pH 5.5 and passed over the
column at a flow rate of 3 ml/h. The column was then washed with the
equilibration buffer followed by the equilibration buffer containing 1 M NaCl, and the enzymes were finally eluted with 20 mM sodium acetate buffer, pH 5.5, containing 5 mM CaCl
, 2.5 M NaCl, and 20% (v/v) propan-2-ol.
All enzyme activities were stable in this buffer for several months
when stored at -20 °C. Enzyme purity was checked by SDS-PAGE
using 12% slab gels. Protein concentration was determined following the
method of Bradford(19) .
Assay for Enzymatic Activity
Enzymatic activity
was normally assayed at 37 °C in a total volume of 100 µl of 50
mM HEPES, pH 7. 0, containing 5 mM CaCl,
with 50 nM [
H]leucine enkephalin as a
substrate(20) . Reactions were stopped by the addition of 10
µl of 0.5 M HCl and the metabolite,
[
H]Tyr-Gly-Gly, was separated for liquid
scintillation counting, using columns of Poropak Q as described
previously(21) . Inhibitors, at varying concentrations, were
preincubated with the enzymes for 30 min before the addition of
substrate, and as the concentration of substrate used for inhibition
and pH studies was less than its K
for
the enzyme, IC
values were taken to be equal to K
values(22) . For pH studies, the
buffers were used at 20 mM with the ionic strength kept at
0.05 with NaCl. K
and k
values for leucine enkephalin degradation were determined using
the substrate over a concentration range of 0.05-4 mM,
with 25 nM [
H]leucine enkephalin
included as a tracer. The values were calculated by linear regression
analysis using the program Enzfit (Biosoft). The change in the free
energies of binding were calculated from
G = RTln((k
/K
)wt/(k
/K
)mut)
or
G = RTln(K
wt/K
mut). In
all cases, reactions were stopped when substrate degradation was
10%.
SDS-PAGE and Western Blots
SDS-PAGE was carried
out using 12% polyacrylamide slab gels. Monoclonal antibodies were
raised against commercially available thermolysin, and one, monoclonal
antibody T1, was found, in enzyme-linked immunosorbent assays, to also
cross-react with NPr. Mapping experiments (not shown) showed that
monoclonal antibody T1 recognizes an epitope contained within residues
206-255 of thermolysin, a region where there are only two amino
acid differences in the sequences of thermolysin and NPr(2) .
For Western blots, 1 ml of the culture was centrifuged at 12,000
g, and 5 µl of the supernatant were mixed with 15
µl of SDS-PAGE sample buffer. For intracellular extracts, the
pellet was resuspended in 100 µl of 50 mM Tris-HCl, pH
7.2, containing 15% (w/v) sucrose and 1 mg/ml lysozyme. After a 5-min
incubation at 37 °C, 180 µl of sample buffer was added, and 10
µl were taken for SDS-PAGE. Electroblotting, to nitrocellulose
sheets, was carried out for 2 h at 200 V, and the blots were developed
using a streptavidine-alkaline phosphatase system (Amersham Corp.).
Expression and Purification of the Wild-type and
Mutated Enzymes
Figure 2:
SDS-PAGE (12%) of purified wild-type and
mutant enzymes. Lane1, 3 µg of wild-type NPr; lane2, 5 µg of H231F-NPr; lane3, 5 µg of H231A-NPr.
Figure 3:
Western blots of extracellular and total
cell extracts of B. subtilis. Extracellular extracts were as
follows: lane1, wild-type NPr; lane2, H231F-NPr; lane3, H231A-NPr. Total
cell extracts were as follows: lane4, wild-type NPr; lane5, H231F-NPr; lane6,
H231A-NPr.
Enzymatic Activity of Wild-type and Mutant Enzymes
values of the substrate, leucine enkephalin for H231F and
H231A-NPr, were almost identical to its K
for the wild-type enzyme, indicating that mutating His-231
does not provoke any major changes in ground state binding. For the
mutant enzymes, however, k
values were lower,
leading to decreases in k
/K
of 430- and 500-fold for H231F and H231A, respectively () equivalent to a change in
G of
3.7-3.8 kcal/mol. These results are comparable with those
obtained with the zinc exopeptidase, carboxypeptidase A (EC 3.4.17.1),
whose active site has a similar topology to
thermolysin(6, 25) . A similar mechanism of action has
been proposed for both enzymes, with Arg-127, corresponding to His-231
of NPr, stabilizing the transition state, and site-directed mutagenesis
of this residue led to large reductions in k
with relatively small changes in K
(26) . In this instance, it was
calculated that the arginine could stabilize the transition state by
4.1-6.0 kcal/mol. Another zinc endopeptidase, neutral
endopeptidase 24.11 (EC 3.4.24.11) is also thought to have an active
site organization that closely resembles that of the Bacillus enzymes, although no crystal structure is available (for review,
see Ref. 7). In this enzyme, His-711 has been proposed to stabilize the
transition state(27) , and mutating this residue to Phe led to a
40-fold reduction in k
/K
, lower than
found here, but the changes were again primarily due to a decrease in k
(28) , indicating that His-711 of the
mammalian enzyme and His-231 of the Bacillus enzymes may have
similar roles in their respective enzymes.
PH Optimum
H]leucine enkephalin by the wild-type enzyme was
in the range of pH 6.0-6.8 (Fig. 4), and, as previously
reported for thermolysin(29) , enzymatic activity varied with
the buffer used, being higher in HEPES than in Tris. For the mutant
enzyme H231F-NPr, optimum activity was between pH 7.5 and 8.5, and at
pH 8.5 in Tris buffer the mutant enzyme was only 30-fold less active
than the wild-type enzyme. The activity of the Bacillus neutral proteases has been shown to depend on two groups, one with
an acidic pK
, suggested to be either
Glu-143 (6) or the zinc-bound water molecule(9) , and a
second species with a pK
around
7.5-8.0, suggested to be His-231 (6, 9). As expected, therefore,
little pH dependence was observed in the alkaline pH range with
H231F-NPr, and the pK
of the remaining
ionizable group was also apparently influenced by the mutation.
Figure 4:
Activity of 0.2 ng wild-type NPr (top) and 50 ng of H231F-NPr (bottom) with pH. The
assay was carried out at constant ionic strength, using 50 nM [H]leucine enkephalin as substrate as
described under ``Experimental Procedures.'' The buffers used
were MES (
), HEPES, (
), and Tris
(
).
Inhibition of Wild-type and Mutant Enzymes
H]leucine enkephalin
hydrolysis by wild-type and mutated enzymes was compared using
representative molecules of the different classes of inhibitors for
neutral proteases (). The presumed binding modes of these
molecules to the active site of NPr are shown in Fig. 5, taken
from data obtained by the cocrystallization of these molecules or their
analogues with thermolysin(6, 30) . All of the molecules
tested have a hydrophobic residue to fit into the S`
subsite and a strong zinc-chelating group. It is important to
note that the K
values of the inhibitors
for the wild-type NPr were not significantly different to those
obtained with thermolysin (31),
(
)confirming the
similarities in the active sites of the two proteases. The values
reported in were obtained using 50 mM HEPES at
pH 7.0 containing 5 mM CaCl
.
Figure 5:
Presumed binding modes of the inhibitors
used in this study to the active site of NPr. Only the expected
interactions with the zinc atom (striatedlines) and
His-231 and Tyr-157 (dottedlines) are shown. A, thiol inhibitors (30, 32); B, hydroxamate inhibitors
(33); C, carboxylate inhibitors (25, 34); D,
phosphoramidon (35).
Thiol Inhibitors
Mutating His-231 to either Phe or Ala
did not modify significantly the K values
of the sulfydryl-containing inhibitors thiorphan, retrothiorphan, and
JCH 24. This is in agreement with molecular modelling studies (20) and the cocrystallization of thiorphan and retrothiorphan
with thermolysin (30), showing that these molecules bind as substrate
analogues, with no apparent interaction with His-231, as previously
reported with another thiorphan-like inhibitor,
(2-benzyl-3-mercaptopropanoyl)-L-alanylglycinamide (32). It
was found that, as predicted(20) , both thiorphan and
retrothiorphan have similar interactions in the active site despite the
retroinversion of the amide bond in the latter. The most important of
these are the coordination of the SH group with the zinc atom, the
binding of the P`
residue in the S`
subsite,
and hydrogen bonding between the carbonyl and carbonyl amide group of
the peptide bond with Arg-203 and Asn-113, respectively. The fact that
mutating His-231 made little difference to the K
values of these inhibitors indicates that these interactions
can still be fulfilled and again shows that the overall geometry of the
active site in its ground state has probably not been greatly altered.
Again these results can be compared with those found for
carboxypeptidase A, where mutating Arg-127 had only a small effect on
the binding of ground state inhibitors(26) .
Hydroxamate and Carboxylate Inhibitors
Mutating
His-231 decreased significantly the affinities of the hydroxamate
inhibitors HACBOGly and ST 51, and the carboxylate inhibitor ES 92. For
HACBOGly, K values were increased
80-fold, and for ST 51 the increases were 100-fold for H231F-NPr and
180-fold for H231A-NPr, equivalent to a change in
G of
between 2.7 and 3.2 kcal/mol. Similar hydroxamate inhibitors have been
described as mimicking reaction intermediates, binding to the active
site of thermolysin in a bidentate fashion, with the carbonyl oxygen
and the hydroxyl oxygen of the hydroxamate group ligated to the
zinc(33) , resembling the presumed pentacoordinate complex of
the transition state in catalysis(6) . The formation of a
hydrogen bond between the carbonyl oxygen of the bidentate group and
His-231 was also predicted. With the carboxylate inhibitor ES 92,
mutating His-231 provoked a 250-fold increase in K
or a change in
G of 3.4 kcal/mol. Again, this
is in line with x-ray crystallographic studies of thermolysin, showing
that the bidentate mode of binding of a carboxylate group resembles the
presumed geometry of the tetrahedral transition state with a hydrogen
bond being formed between one oxygen of the carboxyl group and
His-231(25, 34) .
Phosphoramidon
The largest differences in K values were found with the
phosphoramidate inhibitor, phosphoramidon, which had a K
of 63 nM for the wild-type
enzyme, with no inhibition of either H231F or H231A-NPr being observed
at an inhibitor concentration of 1 mM, giving a difference in
binding energy of >6 kcal/mol. Such a large increase in K
was perhaps unexpected, although
phosphoramidon has been described as a transition state inhibitor with
one of its phosphoryl oxygens ligated to the zinc in a tetrahedral
complex and also forming hydrogen bonds with His-231 and
Tyr-157(6, 35) . In addition, the binding of
phosphoramidon in the alkaline range has been shown to be critically dependent on one ionizable group (36), which is likely to be
His-231. Moreover, mutating Arg-127 of carboxypeptidase A also led to
large decreases in the binding affinity of a transition state analogue
of 5.8 kcal/mol(26) .
Conclusions
, was of the same order as that obtained when
the residue proposed to stabilize the transition state of
carboxypeptidase A (Arg-127) was mutated(26) . This is in
contrast to mutations of Glu-143 from the neutral proteases of B.
stearothermophillus MK232 (37) and B.
subtilis(24) , which led to enzymes with undetectable
proteolytic activity. Similar results were obtained when the
corresponding consensus sequence glutamate of neutral endopeptidase
24.11 was mutated(38) . It was suggested that loss of activity
provoked by these mutations might be due to major perturbations of the
active site, resulting from the replacement of the negatively charged
Glu(9) . However, inactive mutants were obtained even when the
Glu was changed to Asp(37, 38) , and, in addition,
inhibitor binding to the Asp mutant of neutral endopeptidase-24.11 was
unchanged(38) .
values of three thiol inhibitors for
the mutated enzymes. Nevertheless, in spite of the lack of these
stabilizing interactions, such molecules can be potent inhibitors
whatever the type of zinc metallopeptidase
studied(7, 47) . This could be due to favorable
interactions of electrons in the external d orbital of sulfur
with the zinc cation, which does not impose a strict directionality.
For the hydroxamate and carboxylate molecules studied here, an
interaction with His-231 would be expected from x-ray crystallographic
data(6, 33, 34) , and this again was reflected
in the results obtained, with increases in K
values ranging from 80- to 250-fold for the mutated enzymes.
Table: Kinetic constants for the hydrolysis of leucine
enkephalin by wild-type, H231F, and H231A NPrs
Table: K values of inhibitors,
for wild-type and mutated NPrs
-rhamnopyransoyl-(oxyhydroxyphosphinyl)]-L-leucyl-L-tryptophan;
PAGE, polyacrylamide gel electrophoresis; MES,
2-(N-morpholino)ethanesulfonic acid.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.