Additional data about thermolysin specificity in buffer- and glycerol-containing media.

Abstract

Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recognition and elucidation of several new criteria affecting enzyme specificity. Nature and position of the recognized residue, role of adjacent amino acids, lateral chain hydrophobicity, and volume and length of peptides were all considered. Hydrolysis reactions were also carried out in the presence of glycerol; the effect of microenvironment modifications was quantitative, for example in inducing variations in catalytic reaction rates, and also qualitative, such as in influencing affinity.

Full text

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Biochimica et Biophysica Acta 1337 1997 143–148
Additional data about thermolysin specificity in buffer- and
glycerol-containing media
Thierry Lignea,1, Emmanuel Pauthea, Jean-Pierre Montib, Gilles Gacelb,
´
Veronique Larreta-Gardea,)
´
aLaboratoire de Technologie Enzymatique, URA 1442 CNRS, Compiegne UniÕersity, B.P. 529, 60205, Compiegne cedex, France
bFaculte de Pharmacie, 1 rue des LouÕels, 80037, Amiens, France
´
``
Received 2 August 1996; accepted 13 August 1996
Abstract
Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recognition and elucidation
of several new criteria affecting enzyme specificity. Nature and position of the recognized residue, role of adjacent amino
acids, lateral chain hydrophobicity, and volume and length of peptides were all considered. Hydrolysis reactions were also
carried out in the presence of glycerol; the effect of microenvironment modifications was quantitative, for example, in
inducing variations in catalytic reaction rates, and also qualitative, such as in influencing affinity.
Keywords: Metalloproteinase; Enzyme specificity; Thermolysin; Selectivity; Glycerol
1. Introduction
Proteinases are good models for studying the intri-
cate molecular interactions which determine enzyme
w x
specificity1because they recognize particular
cleavage bonds belonging to an infinity of substrates
and not a single substrate.
Metalloproteinases are involved in many important
physiological and industrial processes. They all pos-
sess an essential zinc atom and have similar catalytic
w x
mechanisms 2 , but display considerable differences
in terms of their substrate specificity. Thermolysin
Abbreviations: NMR, nuclear magnetic resonance; TFA, tri-
fluoro acetic acid; RP-HPLC, reverse-phase high-performance
liquid chromatography.
)Corresponding author. Fax: q33 44203910.
1T.L. and E.P. contributed equally to the manuscript.
Ž
of great interest as it displays a structural and func-
tional mimicry and a high degree of active-site ho-
mology such as many microbial and mammalian
metalloendopeptidases — e.g., neutral proteinases
from Bacillus stearothermophilus 4 , angiotensin-
Ž
converting enzyme EC 3.4.15.1
Ž
dopeptidase 24.11 EC 3.4.24.11
Thirty years ago, in pioneer reports, thermolysin
specificity was investigated using proteins and natu-
w x
ral peptides 7 or synthetic ones displaying protect-
w x
ing groups 8 . The enzyme was then described as a
true endopeptidase, except when the terminal residue
bore a hydrophobic protecting group. Thermolysin
recognizes amino group on residues displaying a
bulky and hydrophobic side chain, in the order Ile)
w x
Leu)Val)Phe 9 . The enzyme has a known tridi-
wx
mensional structure, 10 because of this, positioning
. w x
EC 3.4.24.273 , the usual model of the family, is
w x
. w x
. w
5 and neutral en-
x
5,6 .
0167-4838r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.
Ž.
PII S0167-4838 96 00142-2

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T. Ligne et al.rBiochimica et Biophysica Acta 1337 1997 143–148
´
144
wx
of the substrate can be proposed 11 . This implicates
several sub-sites and depends on up 3 amino-acid
residues contiguous to the cleavage bond 8 . The
mechanism of the enzyme has been described on this
wx
basis 12–14 , and refined with the help of ther-
molysin-inhibitor complex structures 15–17 . Sensi-
tive analytic methods and peptide synthesis now al-
low unambiguous determination of the actual speci-
ficity of proteinases. Systematic studies would also
yield new extensive information at the molecular
level about proteolytic process permitting comparison
of various thermolysin-like proteinases.
Moreover, in non-conventional media, modifica-
tions in enzyme specificity have been observed with
various enzymes including proteinases 18,19 . Bound
water molecules play a determining role on affinity
wx
and specificity 20,21 , so that in sugar- or polyol-
containing media, variations in enzyme selectivity
may be observed. These water-restricted media could
provide an additional tool in characterization of sub-
strate preferences and proteinase–substrate interac-
tions.
In this paper, an investigation is performed into the
actual specificity of thermolysin by development and
use of peptide substrates of various lengths in their
natural form, without protecting groups. The study
concentrates on the sensitivity of peptide hydrolysis
to modifications of the reaction medium. Attention
has also been focused on the variations in activity
during peptide hydrolysis in relation to their composi-
tion as well as on the variations in the enzyme
selectivity in order to elucidate enzymersubstrate
recognition.
w x
wx
wx
2. Materials and methods
2.1. Chemicals
Ž.
Thermolysin EC 3.4.24.27 was purchased from
Ž
Sigma Proteinase type X from Bacillus thermopro-
.
teolyticus rokko .
Peptide substrates were designed so as to present
several feasible adjacent cleavage bonds for ther-
molysin in the amino-terminal sequence Gly-Phe,
.
Phe-Ser, Ser-Ala .
hydrophobicrhydrophilic residues according to hy-
wx
dropathy index 22 was chosen so that the peptides
Ž
Thedistribution of
would be soluble in water. The lengths were deter-
mined to allow some 3D-structuration according to
helix propensities of the individual amino acids 23 .
They were synthesized in solid phase using a LKB-
Biochrom 4170 synthesizer with the sequences:
Peptide A: H-Gly-Phe-Ser-Ala-Lys-Asn-Gln-Ser-
OH
Peptide B: H-Gly-Phe-Ser-Ala-Lys-Asn-Glu-Ser-
Asn-Gln-Arg-OH
A tetrapeptide corresponding to the amino-terminal
sequence of peptides A and B H-Gly-Phe-Ser-Ala-
.
OH was produced manually using a chemical syn-
thesis in liquid phase. Tripeptides presenting an amino
terminal Gly, followed by 2 among the 3 next residues
Ž
in the tetrapeptide H-Gly-Phe-Leu-OH, H-Gly-Phe-
Ala-OH,H-Gly-Phe-Ser-OH and H-Gly-Ser-Ala-
..
OH were purchased from Bachem.
wx
Ž
2.2. Hydrolysis reaction
Hydrolysis was performed at 378C in 0.05 M
Ž.
Tris-HCl buffer pH 8.0 . When indicated, glycerol
was dissolved at concentrations ranging from 0 to 10
M in the buffer prior to the addition of substrate. The
reaction medium water activity
from 1 to 0.32 corresponding to 55 to 16 M water
concentrations. It has been checked that addition of
glycerol did not induce any significant pH change.
Usually, the reaction was initiated by addition of
enzyme. Enzymersubstrate ratio was settled to
1r5000 for peptides A and B and to 1r450 for tetra-
and tripeptides. The reaction was stopped by a pH
Ž
drop addition of TFA 0.1% .
Ž.
a
thus ranged
w
.
2.3. Product analysis
Hydrolysis products were analyzed using a RP-
HPLC method with a C18 mBondapak column inter-
nal diameter 3.9 mm, length 30 cm, phase diameter
˚.
10 mm, porosity 125 A . A peptidic bond was re-
vealed at 210 nm.
Elution gradients were obtained with two solu-
tions: solution 1: 0.1% TFA in reagent-grade water;
solution 2: 0.1% TFA in acetonitrile.
Flow rate: 1 ml miny1, sensitivityF4 mM.
Gradients were 0–2 min: 100% solution 1 for all
peptides, then from 2 to 23 min: 60%, 70%, 80%,
Ž
95.3% and 79.6% solution 1 for H-Gly-Phe-Leu-
Ž
Ž.
.

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T. Ligne et al.rBiochimica et Biophysica Acta 1337 1997 143–148
´
145
OH, H-Gly-Phe-Ala-OH, H-Gly-Phe-Ser-OH, Pep-
tide A and Peptide B, respectively.
For H-Gly-Ser-Ala-OH: solution 1: 0–8 min:
100%; 8–13 min 10%.
For H-Gly-Phe-Ser-Ala-OH: solution 1: 0–2 min:
100%; 2–23 min 70%.
These gradients were followed by 2 min of rinsing
Ž.
with 10% solution 1 q90% solution 2 and 10 min
Ž
equilibration with 100% solution 1 .
When two or more products are formed in parallel
reactions, each one is independly measured, allowing
the determination of kinetic constants for each cleav-
age. The products were collected and lyophilized for
further characterization. Amino-acid determination
was performed with PITC derivatization and HPLC
Ž
analysis Waters Picotag devices . Mass spectroscopy
was carried out on a Finnigan SSQ 710 equipped
withan Electrospray
methanolrwaterracetic acid
mixture. NMR was performed on a Brucker-300 MHz
spectrometer.
Ž.
.
.
Analytica
Ž
sourceina
.
50r50r0.5, vrvrv
3. Results and discussion
3.1. Determination of thermolysin specificity
3.1.1. Peptide A and B hydrolysis
Peptides A and B present the same theoretical
Ž
cleavage bonds Table 1A . According to the litera-
.
ture: Ser3-Ala4should be hydrolyzed preferentially
w x Ž
7Ala hydropathy index: 1.8 , Phe -Ser
w x
cleaved 7 but only very slowly or at a high en-
Ž
zymersubstrate ratio Ser hydropathy index: 0.8 ,
Gly-Phe, theoretically considered as a well recog-
Ž
nized bond Phe hydropathy index: 2.8 , should not
be cleaved as it implicates a terminal residue without
w x
a protecting group 9 .
In buffer, Phe2-Ser3and Ser3-Ala4were both
cleaved from the beginning of the reaction. Further-
more contrary to what was expected, Phe2-Ser3was
Ž
cleaved preferentially Table 1B .
.
23
might be
.
.
.
3.1.2. Tetrapeptide hydrolysis
The tetrapeptide behaved as a good substrate for
thermolysin. The previously described hydrolysis of
Ser3-Ala4was not observed with the tetrapeptide.
Two bonds were cleaved by thermolysin, Phe2-Ser3-as
on peptides A and B, and Gly -Phe
Phe2-Ser3bond was cleaved preferentially and this
cleavage represents 70% of the hydrolysis products in
Ž
steady state i.e., complete hydrolysis in 5 h . As
mentioned above the hydrolysis of the two bonds
were independent.
12Ž.
Table 1B . The
.
3.1.3. Tripeptide hydrolysis
Tripeptides were chosen so as to present an amino
terminal Gly, followed by 2 among the 3 next residues
in the tetrapeptide. A comparison was established
with a tripeptide containing Leu which has been
Table 1
Theoretical A and observed B cleavages of synthetic peptides by thermolysin. bold-type arrow priority cleavage, ≠ secondary
cleavage
Ž .Ž .Ž.Ž .

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T. Ligne et al.rBiochimica et Biophysica Acta 1337 1997 143–148
´
146
wx
described as a good marker for thermolysin 7,8 .
w x
According to literature 9 , the hydrolysis of tripep-
tides by thermolysin is unlikely.
The results obtained here confirm 9
longer the peptide, the higher the rate of hydrolysis
Ž
by thermolysin Table 1C . Contrary to what was
w x
previously assumed 9 however, thermolysin dis-
plays an exopeptidase activity Table 1B as homolo-
gous proteinase from Bacillus stearothermophilus
able to cleave the Gly1-Ile2bond in insulin A chain
wx
24 .
The recognition of one bond is not permanent. For
example: Ser-Ala was cleaved in peptides A and B,
but not in the tri- or tetrapeptide, although Ala is a
good marker for thermolysin 8 .
Selectivity in fact concerns the environment of the
cleavage bond. Hydrophobicity is favorable for
recognition of the bond by thermolysin: Gly-Phe-Leu
presents a second cleavage site and the difference
between Gly-Phe-Ser and Gly-Phe-Ala hydrolysis is
due to an additional hydroxide group.
The differences between hydrolysis of various sub-
strates may also be investigated through rate con-
stants. Thermolysin selectivity was obvious from the
variations in K
values, indicating different affini-
m
ties of the enzyme for the different peptide bonds. In
each case, the kinetics followed the Michaelis–
Ž.
Menten model Table 2 . As previously observed,
rate constants show that Gly-Phe-Ala is a better
substrate than Gly-Phe-Ser and that Gly-Phe-Leu is a
w x
that the
.
Ž.
w x
quite good substrate for hydrolysis of Gly1-Phe2but
not for cleavage of Phe2-Leu3.
3.2. Influence of glycerol on thermolysin specificity
3.2.1. Peptide A and B hydrolysis
Addition of glycerol to the reaction medium
changed the hydrolysis kinetics. At glycerol concen-
tration greater than 0.5 M, the rate of hydrolysis of
Ž
peptide A was reduced Fig. 1A and the cleavage of
the Ser3-Ala4bond was delayed by 30 min. Glycerol
also favored one cleavage over two as the ratio
between the various products was changed depending
on glycerol concentrations Fig. 1A . Even a small
amount of polyol affected the reaction rate through
the decrease of Phe2-Ser3cleavage.
For peptide B, the influence of glycerol was less
important, no reaction delayed was observed also no
qualitative difference was observed between either of
the cleavage bonds up to 5 M glycerol Fig. 1B .
Glycerol therefore appears to modify the selectivity
of thermolysin more for peptide A than peptide B.
.
Ž.
Ž.
3.2.2. Tetrapeptide hydrolysis
When 5 M glycerol was added to the hydrolysis
medium of Gly-Phe-Ser-Ala, the cleavage priority
order was not modified. For both hydrolyses, the
reaction rate increased twofold
affinity decreased by an order of magnitude, therefore
resulting in a decreased efficiency.
Ž.
Table 2and the
Table 2
Kinetic constants for each cleavage by thermolysin upon several tripeptides and a tetrapeptide
wx Ž .
SubstrateProduct glycerol M
GFSFS0
5
y13
y1
y1
Ž.Ž.Ž.
k
min
K
mM
k
rK
10 M min
cat
2.1
3
mcat
1.6
0.9
m
1.3
3.5
GFAFA0
5
3.6
4.6
0.3
0.6
12
7.7
GFLFL0
5
0
5
2.7
1.6
0.8
0.4
0.4
1.8
0.7
2.5
6.8
0.9
1.1
0.2
GF
GFSA GF0 18
35.5
4.2
10.4
1.2
12
0.7
6.8
15
3
6
1.5
5
0
5
FSA

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T. Ligne et al.rBiochimica et Biophysica Acta 1337 1997 143–148
´
147
Fig. 1. Influence of glycerol on hydrolysis rate after a 2 hour
Ž.Ž
reaction B and on the ratio MrM between products due to
the Phe-Ser and Ser-Ala cleavages respectively Phe-SerrSer-Ala;
.Ž .Ž .
v ; for A Peptide A and B Peptide B.
.
Ž
3.2.3. Tripeptide hydrolysis
Glycerol influence was mainly sensitive for addi-
tive concentrations over 2 M Fig. 2 . Even in pres-
ence of 5 M glycerol, the kinetics followed a
Michaelis–Menten model and kinetic constants were
Ž.
calculated Table 2 . Depending on the residue next
to the one recognized by the enzyme, the efficiency
of thermolysin to cleave Gly1-Phe2bond was af-
fected differently by glycerol Fig. 3 . For Gly-Phe-
Leu, glycerol affected both cleavages by the same
Ž.
amount Table 2 .
While for high additive concentrations i.e., G2
.
M , the effects observed could be due to modifica-
tions in physico chemical properties of the medium
wx
25 , for low polyol quantities, modifications at the
molecular level should be taken into consideration.
Subtle conformational changes of an enzyme in pres-
ence of polyols have already been described, which
induce dramatic changes in the enzyme behavior 26 .
With tripeptides, glycerol modified the enzyme struc-
Ž.
Ž.
Ž
wx
Fig. 2. Influence of glycerol on hydrolysis rate of Gly-Phe-Ala
Ž.Ž.
` and Gly-Phe-Leu I by thermolysin. Relative effect corre-
sponds to the increasing of half-hydrolysis time. Reference half
hydrolysis times are 310 min and 480 min, respectively.
ture andror reactant diffusion; for larger peptides, a
slight conformational variation of the substrate also
occured as shown by the difference between peptides
A and B. Changing slightly the composition of tripep-
tides, also induced different behavior hydrolysis ac-
.
tivity and selectivity of the enzyme towards these
substrates, showing that the interactions between the
active protein, its substrate and the reaction medium
must be considered at a molecular scale.
The effect of glycerol was also dependent on the
substrate. The influence exerted either on the cat-
alytic rate which is a quantitative variation or on
affinity which is a qualitative variation. The macroef-
fects induced on proteins by the cosolvents — such
as activity lowering — are the direct consequence of
their preferential interactions with the proteins 27 .
Thermodynamically, a change in the standard free
Ž
wx
Fig. 3. Catalytic efficiency of Gly1-Phe2cleavage for different
Ž.
substrates in absence B and presence of glycerol 5 M I .
Catalytic efficiency is expressed as 103My1miny1.
Ž.