ArticlePDF Available

Catalysis by human leukocyte elastase: III. Steady-state kinetics for the hydrolysis of p-nitrophenyl esters

Authors:
Ross Stein at Loxo Oncology, Inc.
Ross Stein
Download full-text PDF
Read full-text
Download citation

Abstract

Steady-state kinetic parameters were determined at pH 7.4 and 25 degrees C for the human leukocyte elastase-catalyzed hydrolysis of several N-carbobenzoxy-L-amino acid p-nitrophenyl esters. The substrate specificity for these esters was quite broad, and included the Gly, Phe, and Tyr derivatives. Together with reports of a much narrower P-1 specificity for peptide-based substrates, these results suggest that interactions remote from the scissle bond between enzyme and substrate regulate primary specificity. Also, it was found that kc and kc/Km did not exhibit the same dependence on substrate structure. This is interpreted to suggest that there are significant differences in P-1 specificity between acylation and deacylation for leukocyte elastase-catalyzed reactions.
Content uploaded by Ross Stein
Author content
All content in this area was uploaded by Ross Stein on Oct 07, 2021
Content may be subject to copyright.
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 236, No. 2, February 1, pp. 677-680, 1985
Catalysis by Human Leukocyte Elastase: III. Steady-State Kinetics
for the Hydrolysis of p-Nitrophenyl Esters’
ROSS L. STEIN
Pulmonary Pharmacology Section, Department
of
Biomedical Research, Stuart Pharmaceuticals,
Division
of
ICI Americas, Wilmingtmz, Delaware 19897
Received August 6, 1984, and in revised form September 28, 1984
Steady-state kinetic parameters were determined at pH 7.4 and 25°C for the hu-
man leukocyte elastase-catalyzed hydrolysis of several N-carbobenzoxy-L-amino acid
p-nitrophenyl esters. The substrate specificity for these esters was quite broad, and
included the Gly, Phe, and Tyr derivatives. Together with reports of a much narrower
P-l specificity for peptide-based substrates, these results suggest that interactions
remote from the scissle bond between enzyme and substrate regulate primary
specificity. Also, it was found that k, and kc/Km did not exhibit the same dependence
on substrate structure. This is interpreted to suggest that there are significant
differences in P-l specificity between acylation and deacylation for leukocyte elastase-
catalyzed reactions.
o 19% Academic PEW I~C.
Substrate specificity for the serine pro-
tease human leukocyte elastase has been
probed by several investigators using pep-
tide amides or p-nitroanilides (3-5). In all
these investigations Val at P-1’ and an
extended peptide structure were found to
be required for maximal catalytic effi-
ciency. In this report the exploration of
substrate specificity for HLE3 is extended
to N-carbobenzoxy-L-amino acid p-nitro-
phenyl esters. Unlike peptide-derived sub-
strates, these esters cannot interact with
HLE at remote subsites and thus provide
the opportunity to examine P-l specificity
in the absence of remote interactions.
Furthermore, the study of HLE-catalyzed
i This is Part III in a series. For Parts I and II
see Refs. (1) and (2).
’ The nomenclature for the amino acid residues of
the substrate (P-l, P-2, P-3 . * . P-n) and corre-
sponding protease subsites to which they bind (S-l,
s-2, s-3 * . . S-n) is that of Schechter and Berger (7).
3 Abbreviations used: HLE, human leucocyte elas-
tase; PPE, porcine pancreatic elastase; CT, bovine
cu-chymotrypsin; MeOSuc-, N-methoxysuccinyl-; pNA,
pnitroaniline; ONP, p-nitrophenol; Nva, norvaline;
Abu, a-aminobutyric acid.
ester hydrolysis may yield information
concerning the specificity of deacylation
since it is this step that generally lim-
its k, for the hydrolysis of esters by serine
proteases (8):
0 0
II II
E-OH + R-C-X k+, E-OH:R-C-X
0 0
II II
E-OH:R-C-X s E-O-C-R -%
X
E-OH + RR-OH
SCHEME I
k, = k2k3/(k2 + k3)
PI
Km = Ksk3/(k2 + k3)
PI
K, = (k-1 + k.J/kl [31
kc/Km = k,k.J(k-, + h) [41
This information is, of course, unavailable
from the study of amide hydrolysis since
677 0003-9861/85 $3.00
Copyright 0 1985 by Academic Press, Inc.
All rights of reproduction in any form reserved.
678
ROSS L. STEIN
these reactions are rate-limited by acyl-
ation (8).
EXPERIMENTAL PROCEDURES
Materials. Human leukocyte elastase was pur-
chased from Elastin Products, (Pacific, MO.). The
material was purified from purulent sputum as de-
scribed previously (9) and supplied as a salt-free,
lyophilized powder. Stock solutions of HLE were
prepared in 0.1 M acetate, 0.5 M NaCl, pH 5.5, and
were found to be stable for several days. Concentra-
tion of active enzyme was determined from estab-
lished kinetics for the hydrolysis of MeOSuc-Ala-
Ala-Pro-Val-pNA (1, 9). HLE preparations were
found to be free of contamination by cathepsin G as
determined with the specific substrate Suc-Ala-Ala-
Pro-Phe-pNA (4).
Z-Gly-ONP, Z-Ala-ONP, Z-Val-ONP, Z-Leu-ONP,
Z-Phe-ONP, Z-Tyr-ONP and Suc-Ala-Ala-Pro-Phe-
pNA were obtained from Sigma Chemical Company
(St. Louis, MO.). Z-Nva-ONP and Z-Abu-ONP were
obtained from Mara Specialty Chemicals, Inc. (Phil-
adelphia, Pa.). MeOSuc-Ala-Ala-Pro-Val-pNA was
available from previous studies (1). Buffer salts were
analytical grade from several sources. Acetonitrile
was analytical grade from Aldrich.
Kinetic procedures. Reaction progress was mea-
sured spectrophotometrically by monitoring the re-
lease of 4-nitrophenolate anion at 400 nm. In a
typical experiment, a cuvette containing 2.88 ml
buffer (10 mM phosphate, 500 mM NaCl, pH 7.4) and
20 ~1 enzyme solution was brought to thermal equi-
librium (5-10 min) in a jacketed holder in the cell
compartment of a Cary 210 spectrophotometer. The
temperature was maintained by water circulated
from a Lauda K-2/RD bath. Injection of 100 ~1 of
the appropriate dilution of a substrate stock solution
in acetonitrile initiated the reaction. Absorbances
were continuously measured, digitized, averaged, and
stored in an Apple II microcomputer. Initial velocities
were calculated by a fit of the experimental data to
a linear dependence on time by linear least-squares
analysis. First-order rate constants were determined
for reactions conducted at [S] Q Km (see below) by
iterative fit of the data to the linearized exponential
equation
WA, - A,) = -kobs. t + ln(A, - A,),
where A, is the absorbance at infinite time, A, is
the absorbance at time t, A0 is the initial absorbance,
and koh is the observed first-order rate constant.
The parameters optimized were A,, A, - Ao, and
kobs. Data were collected for no less than three half-
times.
Data analysis. Values of k, and K,,, and their error
estimates were derived from two to three separate
kinetic experiments, where each experiment consisted
of determining initial velocities, in duplicate or
triplicate, at four to six substrate concentrations.
For each kinetic experiment k, and Km were deter-
mined by nonlinear least-squares fit of the initial
velocity data to the Michaelis-Menten equation.
When necessary initial velocities were corrected for
spontaneous substrate hydrolysis before analysis.
Double-reciprocal plots were linear in all cases.
Values of kJK, were determined in independent
experiments from first-order progress curves re-
corded at 0.10 K,,, > [S] > lO[HLE]. Under these con-
ditions Lb. = (kJK,) [HLE].
RESULTS AND DISCUSSION
The steady-state kinetic data of Table
I indicate that HLE possesses a broad
substrate specificity toward simply, N-acyl
amino acid esters, and contrasts markedly
with the more restricted P-l specificity
generally reported for this enzyme (3-6,
10). Studies of the hydrolysis of peptide
amides and anilides suggest an almost
absolute requirement for Val at P-l (3-
6), and results from recent investigations
of peptide-thioester (10) and aza-peptide
ester hydrolyses (ll), while supporting a
more relaxed specificity, still indicate that
peptides with Gly or Phe at P-l are totally
inactive as HLE substrates. Together
these results indicate that the P-l speci-
ficity of HLE is greatly influenced by this
residue’s amino substituent.
The ability of portions of the substrate
distant from the scissle bond to determine
primary specificity presumably originates
TABLE I
HYDROLYSIS OF N-CARBOBENZOXY-AMINO ACID
~NITROPHENYL ESTERS BY LEUKOCYTE ELASTASE’
Amino
acid Km (PM) kc /Km
(mM -’ s-7
GUY 2.9 _+ 0.2 340 f 40 9 + 0.4
Ala 23 f 3 80 f 4 300 2 10
Abu 7.0 k 0.4 4.8 f 0.5 1,600 f 100
Nva 3.2 f 0.3 5.2 f 0.8 570 f 20
Val 2.7 + 0.1 5.6 _+ 0.2 480 f 10
Leu 1.2 * 0.3 3.6 + 0.7 270 f 50
Phe .23 f .06 3.5 + 0.7 65 f 3
Thr 1.2 f 0.4 75 f 9.5 15 + 2
’ 10 mM phosphate, 500 mM NaCl, pH 7.4, 3.3%
acetonitrile; 25 f O.l”C.
HYDROLYSIS OF pNITROPHENYL ESTERS BY LEUKOCYTE ELASTASE
679
in the “communication” that can occur
between remote substrate binding subsites
and the primary binding site (12). During
the hydrolysis of peptide-derived sub-
strates, subsites past S-l are occupied
and, through subtle changes in the three-
dimensional structure of HLE, cause a
narrowing of the enzyme’s “specificity
pocket” (8) (i.e., the side chain binding
region at S-l). However, when HLE inter-
acts with small substrates remote inter-
actions are impossible and the specificity
pocket remains wide. Thus, while HLE is
an efficient catalyst of Z-Phe-ONP hydro-
lysis [(kc/K,)-Val/(&/K,)-Phe = 71, it is
unable to catalyze the hydrolysis of Suc-
Ala-Ala-Pro-Phe-pNA.
Remote interactions between serine
proteases and their peptide substrates
appear to fulfill several functions during
catalysis by these enzymes. Not only do
these interactions enhance catalytic effi-
ciency (12) and enlist operation of the
charge-relay system (1, 13, 14), but as
shown here, regulate P-l specificity.
The results of Table I reveal another
interesting feature of the specificity of
HLE toward small substrates. If defined
as the correlation between substrate
structure and kc/Km, the specificity of
HLE toward N-carbobenzoxy-amino acid
p-nitrophenyl esters is similar to the
specificity the enzyme displays toward
peptide-derived substrates (3-6, 10,ll). In
all cases kc/Km is largest for substrates
having small hydrophobic amino acid res-
idues at P-l. Furthermore, in all substrate
series the substrate with Ala at P-l has
invariably been found to be less reactive
than substrates with Val, Nva, Leu, or
Ple at P-l. On the other hand, if k, rather
than kc/Km is correlated with substrate
structure, the specificity of HLE toward
the esters of this study is different from
the specificity just described. We see that,
in contrast to kJK,, k, values reveal a
preference for Z-Ala-ONP; substrates de-
rived from other amino acids are less
reactive. Similar results were obtained in
a recent study of thiopeptide ester hydro-
lysis (lo), where it was observed that
changing P-l from Ala to Val, Nva, or
Leu resulted in increases in k,.
An appealing explanation for these dif-
ferences in specificity between k, and kc/
Km comes from consideration of the rate-
determining step for serine protease-cat-
alyzed ester hydrolysis (8). Recall that
these reactions generally occur with rate-
limiting acylation (b) in kc/Km and de-
acylation (k3) in kc4 (8). Thus, the obser-
vation of differences between k, and kc/
K,,, in their correlation with substrate
structure suggests that acylation and de-
acylation possess different specificities.
This conclusion is supported by pre-
steady-state kinetics,4 which directly
demonstrated that the magnitudes of b
and k3 responded differently to variations
in substrate structure.
In contrast to the above are reactions
of HLE with amides and anilides, where
it was observed that substrate structural-
dependent changes in reactivity are re-
flected equally in k, and kc/Km (3, 5, 6).
Without exception it was observed that
changing the P-l residue from Ala to Val
(3, 5, 6) or Ile (3) causes increases of
similar magnitude in both kc and kc/Km.
Since amide hydrolysis generally proceeds
with rate-limiting acylation for both kc
and kc/Km (2, 8), the specificity require-
ments of a single reaction, acylation, are
revealed regardless of which steady-state
kinetic parameter is correlated with sub-
strate structure.
Finally, it is interesting to compare the
substrate specificity of HLE with that of
two other proteases that hydrolyze sub-
strates having neutral amino acid residues
at P-l. In Table II are collected relative
values of kc/Km for the HLE-, PPE-, and
CT-catalyzed hydrolyses of N-carboben-
zoxy-L-amino acid p-nitrophenyl esters.
Despite small differences in reaction con-
ditions, it is clear that values of kc/Km
for the reaction of Z-Gly-ONP with the
three proteases are remarkably similar,
and suggest that this minimal substrate
interacts identically with the three en-
zymes. Only when an alkyl group is sub-
’ Unpublished pre-steady-state kinetic data of the
author for the HLE-catalyzed hydrolysis of N-car-
bobenzoxy-L-amino acid pnitrophenyl esters.
680
ROSS L. STEIN
TABLE II REFERENCES
RELATIVE VALUES OF k,/K,, FOR PROTEASE-
CATALYZED HYDROLYSESOF N-~ARBOBENZOXY-
AMINO ACID p-NITROPHENYL ESTERS
1. STEIN, R. L. (1983) J. Amer. Chem. Sot. 105,
5111-5116.
2. STEIN, R. L., VISCARELLO, B. R., AND WILDONGER,
R. A. (1984) J. Amer. Chem. Sot. 106,796-798.
3. ZIMMERMAN, M., AND ASHE, B. M. (1977) B&him.
Biophys. Acta 480, 241-245.
4. NAKAJIMA, K., POWERS, J. C., ASHE, B. M., AND
ZIMMERMAN, M. (1979) J. BioL C&m. 254,
4027-4032.
Protease
Amino acid HLE” PPE” CT”
GUY 1” 1” 1’
Ala 34 18 1.3
Val 55 0.23 33
Leu 31 0.29 40
Tyr 1.7 0.0014 143
’ 10 mM phosphate, 500 mM NaCI, pH 7.4, 3.3%
acetonitrile; 25°C.
bRef. (15); 100 mM phosphate, pH 8.0, 3% aceto-
nitrile; 21°C.
‘kc/K,,, = 8800 M-’ s-‘.
d k,/K,, = 8300 M-' s-l.
‘kc/K,,, = 16,000 M-' s-i.
stituted for the pro-S-hydrogen of Z-Gly-
ONP do these proteases reveal their true
identities. It is even more striking that
the breadth of substrate specificity dis-
played by HLE resembles that of chymo-
trypsin, and shows little similarity to the
very narrow specificity of porcine elastase.
ACKNOWLEDGMENT
I thank Professor Michael S. Matta (Southern
Illinois University, Edwardsville) for his reading of
this manuscript and helpful comments.
5. MCRAE, B., NAKAJIMA, K., TRAVIS, J., AND Pow-
ERS, J. C. (1980) Biochemistry 19, 3973-3978.
6. MAROSSY, K., SZABO, G. C., POZSGAY, M., AND
ELODI, P. (1980) B&hem. Biophys. Rex Cmn-
mun. 96,762-769.
7. SCHECTER, I., AND BERGER, A. (1967) B&hem
Biophys. Res. Commun 27, 157-162.
8. FERSHT, A. (1977) Enzyme Structure and Mech-
anism, pp. 303-312, Freeman, San Francisco.
9. VISCARELLO, B. R., STEIN, R. L., KUSNER, E. J.,
HOLSCLAW, D., AND KRELL, R. D. (1983) Prep.
B&hem 13,57-67.
10. HARPER, J. W., COOK, R. C., ROBERTS, C. J.,
MCLAUGHLIN, B. J., AND POWERS, J. C. (1984)
Biochemistry 23, 2995-3002.
11. POWERS, J. C., BOONE, R., CARROL, D. L., GUPTON,
B. F., KAM, C. M., NISHINO, N., SAKAMOTO, M.,
AND TUHY, P. M. (1984) J. BioL Chem. 259,
4288-4294.
12. KRAUT, J. (1977) Annu. Rev. B&hem. 46, 331-
358.
13. STEIN, R. L., ELROD, J. P., AND SCHOWEN, R. L.
(1983) J. Amer. Chem. Sot. 105, 2446-2452.
14. ELROD, J. P., HOGG, J. L., QUINN, D. M., VENKA-
TASUBBAN, K. S., AND SCHOWEN, R. L. (1980)
J. Amer. Chem. Sot 102, 3917-3922.
15. ASCENZI, P., MENEGATTI, E., GUARNERI, M., AND
ANTONINI, E. (1983) Mol. Cell. B&hem 56,33-
38.
... For HLE, this has been attributed to the significant effect of subsites on acylation rate. In the absence of an extended chain and therefore remote interactions, HLE, unlike PPE, displays a broad specificity at P 1 [71]; for example, Tyr can be tolerated reasonably well. During hydrolysis of an extended chain, subtle changes in the structure of HLE result in a narrowing of the S 1 pocket [69,71]. ...
... In the absence of an extended chain and therefore remote interactions, HLE, unlike PPE, displays a broad specificity at P 1 [71]; for example, Tyr can be tolerated reasonably well. During hydrolysis of an extended chain, subtle changes in the structure of HLE result in a narrowing of the S 1 pocket [69,71]. Extended interactions appear to fulfil several functions: enhancing catalytic efficiency [72]; enlisting operation of the charge-relay system [69]; regulating P 1 specificity [71]. ...
... During hydrolysis of an extended chain, subtle changes in the structure of HLE result in a narrowing of the S 1 pocket [69,71]. Extended interactions appear to fulfil several functions: enhancing catalytic efficiency [72]; enlisting operation of the charge-relay system [69]; regulating P 1 specificity [71]. ...
Selection of human elastase inhibitors from a conformationally constrained combinatorial peptide library
Article
  • Dec 1999
  • Eur J Biochem
A resin-bound cyclic peptide library was constructed based on the sequence of the reactive-site loop of Bowman–Birk inhibitor, a proteinase inhibitor protein. The constrained loop sequence, which incorporates the minimal proteinase-binding motif, was retained throughout the library, but selected residues known to be important for inhibitor specificity were randomised. The approach was used to create a ‘one bead, one peptide’ library with 8000 variants resulting from randomization at three target locations in the sequence (P4, P1 and P2′). This library allows us to examine the degree to which variations in this proteinase-binding motif can redirect activity, as well as providing information about the binding specificity of a proteinase target. Screening this library for binding to human leucocyte elastase identified sequences with a strong consensus, and on resynthesis all were found to act as inhibitors, with Ki values as low as 65 nm. Human leucocyte elastase is known to have a substrate preference for small alkyl chains at the P1 locus, with valine being preferred. However, alanine and not the expected valine was found in 21 out of 23 identified sequences. The remaining two sequences had threonine at P1, a finding that would be hard to predict based on substrate specificity alone. Further analysis of resynthesized peptides demonstrated that valine substitution results in an analogue that is hydrolysed far more rapidly than ones having library-selected P1 residues. Testing of the human leucocyte elastase-selected sequences as inhibitors of porcine pancreatic elastase demonstrates a significant difference in the specificity of the P4 locus between these two proteinases.
View
... 1,2 Regarding the mechanism, the transition of alveolar type II cells into fibroblasts has been reported to cause and/or aggravate PF. 3,4 The present study exhibited the inhibitory effects of Emodin (the chemical structure was shown in Figure S1A), an anthraquinone mainly found in traditional Chinese medicines such as rhubarb, Polygonum multiflorum and Polygonum cuspidatum, 5,6 on PF through suppressing transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition (EMT) within alveolar epithelial cells via Notch signalling. 7 Further investigation on the potential mechanism of Emodin could possibly provide new strategies to treat pulmonary fibrosis. ...
Emodin inhibiting neutrophil elastase-induced epithelial-mesenchymal transition through Notch1 signalling in alveolar epithelial cells
Article
Full-text available
The transition of alveolar type II epithelial cells into fibroblasts has been reported to cause and/or aggravate pulmonary fibrosis (PF), which is characterized by fibroblast proliferation, an enhanced production and accumulation of ECM (extracellular matrix), alveolar wall damage and functional capillary unit loss. Traditional Chinese medicine Emodin has been reported to inhibit TGF-β-induced epithelial-mesenchymal transition (EMT) in alveolar epithelial cells through Notch signalling. In the present study, neutrophil elastase (NE, also known as ELA2) treatment promoted EMT, Notch1 cleavage (NICD/Notch1 ratio increase) and NICD nuclear translocation in RLE-6TN cells and A549 cells. The promotive roles of NE treatment in these events were significantly reversed by Notch1 knockdown. Traditional Chinese medicine Emodin treatment remarkably inhibited the enzyme activity of NE, suppressed EMT, Notch1 cleavage and NICD nuclear translocation within RLE-6TN and A549 cells, while NE treatment significantly reversed the effects of Emodin. Moreover, in RLE-6TN, the effects of NE on EMT, Notch1 cleavage and NICD nuclear translocation were remarkably attenuated by Emodin treatment and more attenuated by the combination of Emodin and neutrophil elastase inhibitor Sivelestat or notch signal pathway inhibitor DAPT. In conclusion, we revealed the involvement of NE-induced Notch1 cleavage in the functions of Emodin suppressing NE-caused EMT in RLE-6TN cells and A549 cells. This novel mechanism of Emodin inhibiting EMT might extend the application of Emodin in PF treatment.
View
A non‐contact device for fast screening of wound infections
Article
  • Jun 2021
Screening for wound infection relies on the expertise of the provider. Clinical diagnosis of infections based on wound swab/biopsy results often takes a few days and may not assess the full wound. There is a need for a non‐invasive tool that can quickly and accurately diagnose wound infection. Leukocyte esterase strips are used to identify various infectious diseases. However, it is not clear whether infected wounds also have elevated leukocyte esterase activities as compared with non‐infected wounds. To achieve the objective, a device was developed to detect elevated leukocyte esterase activities in wounds by measuring wound exudates adsorbed onto wound dressings in 3 minutes. The efficacy of the device in assessing leukocyte esterase activities across various chronic wounds was tested. Such measurements were unaffected by the type of underlying wound dressing. By correlating the device outputs with clinical adjudication of infection, we found that this device had high positive predictive values for diagnosing wound infection in a wide variety of chronic wounds. In addition, a positive device output increases the probability of detecting infected wounds, while the negative device output reduces the probability of detecting infected wounds. This rapid non‐contact and disposable diagnostic tool may serve as a rapid and accurate indication of infection in the chronic wound.
View
Chapter 24. Neutrophil Elastase
Article
  • Dec 1985
  • ANNU REP MED CHEM
This chapter discusses the biochemical properties, roles in normal physiology, and possible involvement in pathological states of the elastase from human neutrophils. Human leukocyte elastase (HLE) is a serine proteinase,as evidenced by its inhibition by diisopropyl fluorophosphate, phenylmethyl sulfonyl fluoride, and alpha-1-proteinase inhibitor. It catalyzes the hydrolysis of substrate esters and amides, according to a mechanism, involving the formation and subsequent hydrolysis of a covalent acyl-enzyme. The importance of human leukocyte elastase as a pathogenic agent in a variety of disease states in man has been recognized. This has resulted in a surge of interest in defining the substrate specificity and designing potent synthetic inhibitors of HLE. α1-Proteinase inhibitor (α1-PI, α1-antitrypsin) is the most abundant serum proteinase inhibitor. In recent years, a variety of synthetic peptidic compounds have been reported as simple competitive and reversible inhibitors of HLE. Ultrastructural, cytochemical, and immunofluorescence studies of human neutrophils have demonstrated the presence of elastase primarily in the azurophilic granules and, also, in the nuclear envelope, golgi complex, endoplasmic reticulum, and mitocondria. In general, proteinases and esterases present in the cytoplasm and membranes of the leukocyte play an important role in the generation of and response to chemotactic stimuli. Elastase is one of the major fibrinolytic proteinases of the leukocyte. Early events in blood coagulation induce the active secretion of elastase from leukocytes. Neutrophil elastase has been implicated as a pathogenic agent in a number of disease states in man, most notably, pulmonary emphysema. Moreover, HLE is thought to play a causative role to the pathogenesis of rheumatoid arthritis, adult respiratory distress syndrome, glomerulonephritis, and cystic fibrosis.
View
Transition-state properties for the association of .alpha.-1-protease inhibitor with porcine pancreatic elastase
Article
Full-text available
  • Oct 1985
The proton inventory (rate measurements in mixtures of H2O and D2O) was determined for the association of human α-1-protease inhibitor (αPI) with porcine pancreatic elastase (PPE). The overall solvent deuterium isotope effect [D(kE)] is 0.91 ± 0.06. As simpler models of this reaction, the proton inventories were also determined for the reaction of PPE with Suc-(Ala)3-pNA [D(kE) = 2.02 ± 0.09] and human leukocyte elastase (HLE) with MeOSuc-Ala-Ala-Pro-Val-pNA [D(kE) = 1.51 ± 0.04]. For all three reactions, the general shape of the proton inventory was dome-shaped, and, in fact, for the association of αPI and PPE, the curve displayed a maxima. These curves could be described by the single expression (Formula Presented) where n is the mole fraction solvent D2O, Z is a composite fractionation factor corresponding to solvent reorganization and general "medium effects", C1 and C2 are the contributions made by each of the two partially rate-limiting transition states of kE, a physical step (C1), and the chemical steps of acylation (C2), Φ2 is a composite fractionation factor corresponding to the transition state for the physical step, and Φ2 is one of two identical fractionation factors corresponding to the two exchangeable protonic sites of the charge-relay system. These experiments suggest that the association of αPI and PPE proceeds through a transition state that derives its stability in part from some sort of protolytic catalysis and may therefore be at least partially rate-limited by acylation. In addition, these results point out the general importance of solvent reorganization in associative processes of enzymic reactions.
View
Catalysis by human leukocyte elastase. Role of secondary-subsite interactions
Article
Full-text available
  • Oct 1985
Steady-state and pre-steady-state kinetic constants, solvent deuterium isotope effects, and proton inventories were determined for the (HLE) human leukocyte elastase-catalyzed hydrolyses of the p-nitrophenyl esters and p-nitroanilides of N-(carbobenzoxy)-L-amino acids and N-(methoxysuccinyl)-Ala-Ala-Pro-Val, an HLE-specific peptide. The results of these experiments indicate that remote subsite interactions between protease and substrate have a significant effect on four mechanistic features: (i) Catalytic Efficiency. Changing the carbobenzoxy group of Z-Val-X (X = p-nitrophenol or p-nitroaniline) to MeOSuc-Ala-Ala-Pro resulted in large increases in both kc and kc/Km and confirmed studies by other investigators documenting the effect that peptide chain length has on catalytic efficiency. Pre-steady-state kinetic experiments further demonstrated that subsite interactions have a greater effect on acylation than on deacylation: while k2 (first-order rate constant for reaction within the Michaelis complex to form the acyl-enzyme) increased 50000-fold upon changing Z-Val-p-nitroanilide to MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide, k3 (first-order rate constant for hydrolysis of the acyl-enzyme) increased only 6-fold, (ii) P1 Specificity. The effect of remote interactions on P1 specificity during deacylation was assessed by comparing ratios of k3 for pairs of substrates having either Ala or Val at P1. k3-Ala/k3-Val varied with substrate structure past P1 as follows: Z-AA, 13; t-Boc-Ala-Ala-AA, 7; MeOSuc-Ala-Pro-AA, 3.5; and MeOSuc-Ala-Ala-Pro-AA, 2. (iii) Rate-Determining Step. kc [=k2k3/(k2 + k3)] was found to be rate-limited by k2 for the p-nitroanilides of Z-Val and other minimal acyl portions. In contrast, very specific peptide-derived anilides which afford the enzyme extensive opportunities for subsite contacts acylate HLE quite efficiently and are hydrolyzed with rate-limiting k3. Furthermore, the process governed by k2 appears to have a "virtual transition state" composed of the transition state for acylation, which is rate-limiting for minimal substrates, and the transition state for some physical step, which becomes partially rate-limiting for specific substrates. (iv) Charge-Relay Catalysis. Despite only a 6-fold difference in deacylation rates between Z-Val-HLE and MeOSuc-Ala-Ala-Pro-Val-HLE, the nature of the protonic catalysis involved in these processes appears to be different. The data are consistent with a mechanism for Z-Val-HLE hydrolysis in which only the active-site His acts as the general catalyst. Hydrolysis of MeOSuc-Ala-Ala-Pro-Val-HLE, on the other hand, proceeds with full involvement of the charge-relay system. The catalytic role of the charge-relay system is discussed in light of these observations.
View
Phytochemische Untersuchungen von zwei Heilpflanzen Zentralamerikas sowie pharmakologisch-biologische Untersuchungen von Naturstoffen mit AP-1, MMPs und Humaner Neutrophiler Elastase als molekulare Targets
Article
  • Jan 2008
1. Die biologisch kontrollierte Fraktionierung eines Extraktes aus Blättern der traditionell verwendeten Euphorbiaceae Hieronyma alchorneoides Fr. Allemão, führte zur Isolierung und Strukturaufklärung (mittels NMR- und MS-Untersuchungen) der drei Biflavonoide 7,4‘‘-O-Dimethylamentoflavon (H.1), 7-O-Methyl-amentoflavon (H.2) und Amentoflavon (H.3) sowie von (+)-Catechin (H.4). Die Verbindungen H.1 und H.2 sowie H.4 sind im Rahmen dieser Arbeit erstmals für die Gattung Hieronyma beschrieben worden. 2. Die phytochemische Untersuchung eines Extraktes aus den Blättern von Siparuna thecaphora (Poepp. et Endl.) A. DC. (Siparunaceae), der im MTT-Test eine starke cytotoxische Aktivität aufwies, führte zur Isolierung des Flavonoids 3,7,4‘ Trimethoxykämpferol (S.1) und des Alkaloids Aristolactam AIIIa (S.2). S.1 ist hier für die Art, S.2 für die Gattung erstmals beschrieben. Zur Strukturaufklärung wurden MS- und NMR-Daten eingesetzt. 3. Es konnte gezeigt werden, dass Sesquiterpenlactone aus den Blüten von Arnica montana sowie Arnikazubereitungen die DNA-Bindung des Transkriptionsfaktors AP 1 in Jurkat-T-Zellen hemmen. Helenalinisobutyrat führte in einer Konzentration von 5 µM zu einer vollständigen Hemmung der IL-1β-induzierten Aktivierung von AP-1. Für den gleichen Effekt wurde von Dihydrohelenalinisovalerianat und Dihydrohelenalinmethacrylat eine Konzentration von ca. 20 µM benötigt. Dies steht im Einklang mit vorhergehenden Analysen mit dem Transkriptionsfaktor NF-κB, die ebenfalls eine stärkere Aktivität von Helenalinderivaten gezeigt haben. In Übereinstimmung damit zeigte eine Tinktur aus mitteleuropäischen (ME), getrockneten Arnikablüten eine deutliche Hemmung der AP-1-DNA-Bindung bei einer Konzentration von 2 µL/mL. Von der Tinktur aus frischen Blüten (ME) und einer Tinktur aus spanischen Arnikablüten waren 5 µL/mL für eine vollständige Hemmung der nach Stimulierung mit IL-1β induzierten Aktivierung von AP-1 erforderlich. Das Gel, das eine Tinktur aus frischen Blüten (ME) enthält, zeigte diesen Effekt bei 10 µL/mL. 4. Weitergehende Untersuchungen zum Wirkmechanismus der AP-1-Hemmung mit Parthenolid und 4β-15-Epoxymiller-9Z-enolid zeigten, dass Sesquiterpenlactone AP 1 direkt angreifen. 5. Die verschiedenen Arnikazubereitungen wurden außerdem auf ihre Hemmaktivität gegenüber der IL 1β-induzierten Expression von MMP1 und MMP13 auf Genebene in Chondrocyten mittels qRT-PCR untersucht. Alle Zubereitungen zeigten eine konzentrationsabhängige, zu der Vergleichssubstanz Helenalinisobutyrat (2 µM) ähnlich starke Verringerung der Genexpression in einem Bereich von 0,2 µL bis 5 µL/mL in bovinen Chondrocyten. Diese starke, konzentrationsabhängige Hemmung konnte exemplarisch auch für Arnikagel in humanen Chondrocyten gezeigt werden. 6. Agrimoniin, Pedunculagin und EGCG konnten als starke direkte Inhibitoren von Humaner Neutrophiler Elastase identifiziert werden. Die IC50 Werte lagen hierbei zwischen 0,9 µM und 25 µM. Sieben weitere phenolische Verbindungen zeigten keine oder nur eine geringe Hemmaktivität. Bei den Untersuchungen, in wieweit die Substanzen die Elastasefreisetzung aus Neutrophilen Granulocyten hemmen können, wurden als aktivste Verbindungen Genistein mit einem IC50-Wert von 0,5 µM sowie Resveratrol mit einem IC50-Wert von 12 µM ermittelt. Alle anderen Substanzen, wie Tyrosol, Hydroxytyrosol, Triacetoxystilben, (+) Epicatechin und (+)-Epicatechin-(4α-8)-(+)-epicatechin, waren schwächer aktiv, die Hemmung lag dort bei einer Konzentration von 50 µM teilweise deutlich unter 50 %. 7. Es wurde ein Elastase-Assay im Arbeitskreis etabliert, um das in Kooperation mit anderen Arbeitskreisen nach Bindungsstudien und chemischer Synthese zur Verfügung stehende Bornyl-3,4,5-trihydroxycinnamat zu testen. Die Aktivität lag mit einem IC50-Wert von 0,54 µM über der der Vergleichssubstanz Bornylcaffeat (IC50-Wert von 1,56 µM). 8. In einem weiteren Kapitel wurde eine Übersicht über die aktuelle Literatur zur Bedeutung von Gerbstoffen bei topischer Anwendung, ihren Einsatzgebieten und möglichen Wirkmechanismen zusammengestellt und diskutiert.
View
Human neutrophil proteinase 3: Mapping of the substrate binding site using peptidyl thiobenzyl esters
Article
  • Dec 1992
A series of peptidyl thiobenzyl esters was used to map the active site of human leukocyte proteinase 3. The steady-state kinetics parameters reveal the following features regarding the substrate specificity of proteinase 3 and its putative active site: (a) the preferred P1 residue is a small hydrophobic amino acid such as aminobutyric acid, norvaline, valine or alanine (in decreasing order of preference); (b) the enzyme has an extended active site; and (c) its active site is similar to that of the related serine proteinases leukocyte elastase and leukocyte cathepsin G.
View
Novel anthraquinone inhibitors of Human Leukocyte Elastase and cathepsin G
Article
  • Jun 1992
A large series of variously substituted anthraquinones has been synthesized and assayed for inhibitory capacity against human leukocyte elastase (HLE) and cathepsin G (CatG), two serine proteinases implicated in diseases characterized by the abnormal degradation of connective tissue, such as pulmonary emphysema and rheumatoid arthritis. It was found that 2-alkyl-1,8-dihydroxyanthraquinone analogues are competitive inhibitors of HLE with IC50 values ranging from 4 to 10 microM, and also inhibit CatG with IC50 values ranging from 25 to 55 microM. Consequently, analogues containing the 2-alkyl-1-hydroxy-8-methoxyanthraquinone substitution pattern inhibit HLE to the same magnitude as for the compounds above, but show very little inhibition of CatG. Anthraquinones containing long, hydrophobic n-butyl carbonate moieties in the 1- and 8-positions in conjunction with a third hydrophobic substituent in the 2- or 3-position are highly selective for HLE, with Ki values in the range of 10(-7) M. All of the inhibitors described are completely reversible, with no evidence of acyl-enzyme formation detected.
View
Enantioselective inhibition of human leukocyte elastase
Article
  • Sep 1992
(RS)-Diethyl-2-benzyl-succinate was resolved using alpha-chymotrypsin. The two enantiomers were then elaborated to yield (S)-(+) and (R)-(-)-3-benzyl-N-[(methyl-sulfonyl)oxy]succinimide and the inhibitory activity of the two enantiomers toward human leukocyte elastase was subsequently determined. The k2/KI values for the R and S isomers were found to be 330 and 1500 M-1 s-1, respectively.
View
Catalysis by human leukocyte elastase: Substrate structural dependence of rate-limiting protolytic catalysis and operation of the charge relay system
Article
Full-text available
  • Jul 1983
The catalytic mechanism of human leukocyte elastase (HLE) has been probed by the determination of solvent isotope effects (calculated as the ratio of rate constants kH2O/kD2O) for the hydrolysis of three substrates of varying reactivity toward HLE: N-succinylalanylalanylalanine p-nitroanilide (I; kc/Km = 450 M-1 s-1), N-succinylalanylalanylvaline p-nitroanilide (II; kc/Km = 4800 M-1 s-1), and N-methoxysuccinylalanylalanylprolylvaline p-nitroanilide (III; kc/Km = 185 000 M-1 s-1). For all three substrates, the isotope effect on kc is near 3 and suggests that the rate-limiting step involves proton transfer. kc [=(k2k3)/(k2 + k3)] is a complex first-order rate constant reflecting both the conversion of enzyme-substrate complex to acylated enzyme (k2) and the subsequent hydrolysis of this intermediate to free enzyme and product (k3). The observed isotope effects on kc for the three substrates are consistent with a mechanism for transition-state stabilization involving some form of general-acid/general-base, or protolytic, catalysis. For substrates I and II the isotope effect on kc/Km is again large and near 2.5. kc/Km is the second-order rate constant for reaction of free enzyme and substrate and can reflect transition-state properties of all steps up to and including the release of the first product, p-nitroaniline. The solvent isotope effects on this process for I and II suggest a transition state involving proton transfer and are consistent with rate-limiting protolytically catalyzed acylation. In contrast, the isotope effect on kc/Km for the specific substrate III is small, equal to 1.4, and suggests that acylation is now only partially rate limiting. The transition state we observe here is a "virtual" transition state, a composite having a structural feature of the several rate-limiting transition states. The small observed isotope effect originates from dilution of a large isotope effect for acylation by a near unit isotope effect for the other partially rate-limiting step(s). Proton inventories (rate measurements in mixtures of H2O and D2O) for the reactions of substrates I and II gave linear dependences of reaction velocity on mole fraction of solvent deuterium, n, and suggest that the isotope effects observed for these reactions originate from fractionation at a single hydrogenic site in the catalytic transition state. Presumably this site is the proton bridge between the active-site serine hydroxyl and histidine imidazole. For the hydrolysis of III by HLE a quadratic dependence of reaction rate on n was observed, suggesting that the isotope effect seen for this reaction originates from coupled transfer of two protons in the catalytic transition state and implying the involvement of a general-acid/general-base functionality beyond the active site histidine. This observation is consistent with a mechanism for HLE in which specific substrates such as III can fully engage the catalytic machinery of the charge-relay system.
View
Correlative variations in enzyme-derived and substrate-derived structures of catalytic transition states. Implications for the catalytic strategy of acyl-transfer enzymes
Article
Full-text available
  • Apr 1983
Acetylchymotrypsin, acetyl elastase and (carbobenzyloxy)glycyl elastase all undergo hydrolysis with the same overall solvent isotope effect, which arises from a single protonic site [kn/k1 = 2.45(1 - n + n/2.45)]. [(Carbobenzyloxy)glycyl]chymotrypsin, however, shows a larger effect arising from at least two sites [kn/k1 = 3.34(1 - n + n/1.85)2]. Formylchymotrypsin and acetylchymotrypsin undergo deacylation with α-deuterium and β-deuterium secondary isotope effects, respectively, that suggest fractional tetrahedral character at the transition state of about 0.44 (vs. 0.58-0.66 for similar nonenzymic reactions) when compared to equilibrium isotope effects for complete addition. The effect for acetyl elastase suggests much less tetrahedral character (0.27). Addition of an N-acyl function leads to a more inverse isotope effect, per deuterium, and thus to an apparent increase in tetrahedral character: to 0.84 for [(carbobenzyloxy)glycyl]chymotrypsin; to 0.43 for (carbobenzyloxy)glycyl elastase. It is concluded that enzyme-substrate interactions at the transition state can alter both enzyme structure, as shown by the solvent isotope effects, and substrate structure, as shown by the substrate isotope effects. Such alterations, in the combination of enzyme with natural substrate, probably adjust both structures for optimal catalytic interaction.
View
View
Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the alpha 1-protease inhibitor reactive site
Article
Full-text available
  • Jun 1979
The kinetic constants for the hydrolysis of a series of 4-nitroanilide substrates by human leukocyte (HL) elastase and cathepsin G, porcine pancreatic elastase, and bovine chymotrypsin at pH 7.50 are reported. HL elastase and cathepsin G are currently thought to be the agents responsible for destruction of the lung in the disease emphysema. MeO-Suc-Ala-Ala-Pro-Val-NA is an excellent substrate for HL elastase and is not hydrolyzed by cathepsin G. The MeO-Suc-group increases the solubility of a substrate relative to the acetyl group. With HL elastase, this structural change increases the reactivity of the enzyme toward both 4-nitroanilide substrates and chloromethyl ketone inhibitors. This indicates that HL elastase is interacting with at least 5 residues of a substrate (or inhibitor). Cathepsin G prefers P 5 groups which are negatively charged such as Suc-, Suc(4F)-, Glt-, or Mal-. This enzyme, in common with many other serine proteases, cannot accept a Pro residue at its S 3 subsite. One of the better substrates for cathepsin G, Suc-Ala-Ala-Pro-Phe-NA, was not hydrolyzed by HL elastase. These tools should be useful in the study of the biological function of HL elastase and cathepsin G. Two tetrapeptide 4-nitroanilide substrates related to the reactive site of the plasma α 1-protease inhibitor (α 1-antitrypsin) were studied. Both have a P 1 Met residue and one, MeO-Suc-Ala-Ile-Pro-Met-NA, has the exact sequence of the P 4 to P 1 residues at the proteolysis site of α 1-PI (Johnson, D.A., and Travis, J. (1978) J. Biol. Chem. 253, 7142-7144). Both MeO-Suc-Ala-Ala-Pro-Met-NA and MeO-Suc-Ala-Ile-Pro-Met-NA react with cathepsin G, HL elastase, and bovine chymotrypsin. The former is in fact the best 4-nitroanilide substrate of cathepsin G yet reported. Oxidation of MeO-Suc-Ala-Ala-Pro-Met-NA yielded two diastereomeric sulfoxides. Neither are bound to or was hydrolyzed by HL elastase or cathepsin G. Both reacted poorly with bovine chymotrypsin. In the preceding paper, Johnson and Travis (Johnson, D., and Travis, J. (1979) J. Biol. Chem. 254, 4022-4026) show that oxidation of α 1-PI destroys its inhibitory activity. In concert, our results indicate that oxidation of the P 1 Met of α 1-PI is capable of destroying its reactivity toward most serine proteases. Oxidation of α 1-PI by some component in cigarette smoke would offer one explanation in molecular terms for the link between smoking and emphysema.
View
Protonic reorganization and substrate structure in catalysis by serine proteases
Article
  • May 1980
Proton inventories (rate measurements in binary mixtures of protium and deuterium oxides) have been used to estimate the number of protons involved in hydrolytic catalysis by serine proteases with various substrates. Trypsin with the oligopeptide analogue BzPhe-Val-Arg p-nitroanilide as substrate produces an overall solvent isotope effect VH2o/ VD2O of 4.30, and a proton inventory consistent with two-proton catalysis, with each proton generating an isotope effect of about 2.1. α-Chymotrypsin with the truncated substrates AcTrpNH2 (acylation rate limiting) and 4-nitrophenyl 3-phenylpropanoate (deacylation rate determining) also produces apparent two-proton catalysis but with smaller overall isotope effects and skewed contributions from the two sites (VH2o/VD2O = 1.90 ∼ 1.69 × 1.14 for AcTrpNH2 and VH2o/VD2O = 2.85 ∼ 1.85 × 1.54 for 4-nitrophenyl 3-phenylpropanoate). On the other hand, trypsin with the similar substrate BzArgOEt (VH2O/VD2O = 3.03) and thrombin with BzArgOEt (VH2O/VD2O = 2.92) give one-proton results, with deacylation presumably rate limiting in both cases. The minimal substrate p-nitrophenyl acetate with α-chymotrypsin (VH2O/VD2O = 2.40) and elastase (VH2O/VD2O = 2.92) shows one-proton catalysis for rate-determining deacetylation, while with trypsin the overall effect (VH2o/VD2O = 1.38) is too small to resolve the question of the number of active protons. Apparently, oligopeptide structure, leading to enzyme-substrate interactions at remote subsites as well as at the catalytic site in the catalytic transition state, is required to bring into action the full evolutionarily developed acid-base machinery of the serine proteases. If the structure is reduced to the point where only catalytic site interactions occur, the reliability of the acid-base machinery is much impaired, while, with minimal substrates, the enzyme acts either as a simple general catalyst or perhaps even as a nucleophilic catalyst. Compression of the distance across the catalytic hydrogen-bond chain of the active site as a consequence of remote-site interactions in the transition state, with relaxation of the enzyme structure occurring in their absence, is a reasonable mechanism for the coupling and decoupling of the protonic interactions by substrate structure.
View
Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study
Article
  • Mar 1983
Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: $${\text{E + S}}\mathop {\mathop \rightleftharpoons \limits_{{\text{k}}_{{\text{ - 1}}} } }\limits^{{\text{k}}_{{\text{ + 1}}} } {\text{E}} \cdot {\text{S}}\mathop {\mathop \rightleftharpoons \limits_{{\text{k}}_{{\text{ - 2}}} } }\limits^{{\text{k}}_{{\text{ + 2}}} } {\text{E}} \cdot {\text{P + P}}_{\text{1}} \mathop {\mathop \rightleftharpoons \limits_{{\text{k}}_{{\text{ - 3}}} } }\limits^{{\text{k}}_{{\text{ + 3}}} } {\text{E + P}}_{\text{2}} .$$ Under pre-steady-state conditions, where [E0] ≫ [S0], the values of the dissociation constant of the E · S complex (Ks = k−1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] ≫ [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 ± 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenylesters of N-α-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.β-trypsin and b.α-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p. p. elastase, b.β-trypsin and b.α-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.
View
View
Substrate specificity of the elastase and the chymotrypsin-like enzyme of the human granulocyte
Article
  • Feb 1977
  • Biochim Biophys Acta
Human granulocyte elastase (EC 3.4.21.11) differs from hog pancreatic elastase in its specificity for synthetic substrates. Although hydrolyzing peptide bonds adjacent to the carboxyl group of alanine, the granulocyte enzyme prefers valine at the cleaved bond, in contrast to the pancreatic enzyme which prefers alanine. Peptide bonds involving the carboxyl group of isoleucine can be hydrolyzed by the granulocyte enzyme but are not hydrolyzed to any significant extent extent by pancreatic elastase. This difference in specificty could explain the lower sensitivity of the granulocyte enzyme to inhibitors containing alanine analogs, such as the peptide chloromethyl ketones and elastatinal. The human granulocyte chymotrypsin-like enzyme differs from pancreatic chymotrypsin by being able to cleave substrates containing leucine in addition to those containing the aromatic amino acids.
View
View
Active site mapping of the serine proteases human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II. Bovine chymotrypsin A alpha, and Staphylococcus aureus protease V-8 using tripeptide thiobenzyl ester substrates
Article
  • Jul 1984
The primary subsite specificities of human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and the protease from strain V-8 of Staphylococcus aureus have been mapped with a series of tripeptide thiobenzyl ester substrates of the general formula Boc-Ala-Ala-AA-SBzl, where AA represents one of 13 amino acids. In addition, the effects of a P2 Pro and P4 methoxysuccinyl and succinyl groups were investigated. In an attempt to introduce specificity and/or reactivity into the substrate Boc-Ala-Ala-Leu-SBzl(X), the 4-chloro-, 4-nitro-, and 4-methoxythiobenzyl ester derivatives were studied. Enzymatic hydrolyses of the substrates were measured in the presence of 4,4'-dithiobis(pyridine) or 5,5'-dithiobis(2-nitrobenzoic acid), which provided a highly sensitive assay method for free thiol. The thio esters were excellent substrates for the enzymes tested, and in many cases, the best substrates reported here have kcat/KM values higher than those reported previously. The best substrate for human leukocyte elastase was Boc-Ala-Pro-Nva-SBzl(Cl), which has a kcat/KM of 130 X 10(6) M-1 s-1. A very reactive rat mast cell protease substrate, Boc-Ala-Ala-Leu-SBzl(NO2), was also found. The S. aureus V-8 protease was the most specific enzyme tested since it hydrolyzed only Boc-Ala-Ala-Glu-SBzl. Substituents on the thiobenzyl ester moiety of Boc-Ala-Ala-Leu-SBzl resulted in decreased KM values with human leukocyte elastase and rat mast cell protease I when compared to the unsubstituted derivative.(ABSTRACT TRUNCATED AT 250 WORDS)
View