Proton Bridges in Thrombin-Catalyzed Hydrolysis of Tripeptide 4Nitroanilides
Linnea Patt
Senior Comprehensive Paper
Catholic University of America
Fall 2003
Abstract:
The purpose of this study was to identify the number of protonic sites and
characterize their role in catalysis by thrombin in the hydrolysis of chromogenic
substrates that contain some of the P1-P3 specificity sites. For N-Benzoyl-Phe-Val-ArgpNA HCl, the solvent isotope effects were found to be: at 25.0 0.1 oC, 2.8 0.2 for the
Vmax calculations, and 1.2 0.2 for the Vmax/KM calculations; and at 37.0  0.2 oC and 1.6
0.1 for the kobs calculations, and 2.21  0.03 for the v calculations. The fractionation
factors were found to be: at 25.0 0.1 oC, 0.37  0.07 for the Vmax calculations, and 0.3 
0.3; 2.3  1.8 for the Vmax/KM calculations, and at 37.0  0.2 oC, 0.58  0.09 for the kobs
calculations, and 0.39  0.04; 1.2  0.1 for the v calculations. The data are most
consistent with having a single proton participate in catalysis.
2
Introduction:
Blood clotting is an important physiological process in vertebrate animals that
works as a defense mechanism to prevent blood loss. Both the formation and breakdown
of clots must occur rapidly in a highly regulated environment to ensure proper blood flow
and function. This regulation is achieved by a highly complex system called an
enzymatic cascade.
Enzymatic cascades are efficient processes in biological systems that ensure a
rapid response to an outside trigger. The response begins with an escalation of biological
function, such as the increase in concentration and activity of certain enzymes. Trauma
and/or rupture to a blood vessel initiates an intrinsic and/or extrinsic pathway that begins
the complex cascade of blood clotting. Once the cascade is started, many zymogens, or
proenzymes, are activated and the signal is amplified greatly. In each highly regulated
step of the process, proteins are cleaved and activated in order to cleave and activate
other proteins in the cascade. Thirteen proteins are activated in the cascade culminating
in the generation of thrombin,1 as seen in Figure 1.
Thrombin is considered the key enzyme in the clotting cascade because it not only
is involved in the last step of the cascade, but it also works to regulate the cascade. To
perform its regulatory functions, there are two forms of thrombin: fast and slow.2 At
adequately high concentrations of Na+ (0.2M), it performs the last step in the process of
conversion of fibrinogen to fibrin monomers. Fibrinogen is made up of three globular
units connected by six chains. The tails of four chains are cut, which allows fibrin
monomers to assemble into a fibrous array called fibrin, which traps platelets and other
blood proteins to form the blood clot.1 Ion fluxes in the blood change throughout the
3
course of blood clotting and eventually the Na+ concentration will drop and the Ca2+
concentration will rise. These conditions enhance thrombin’s affinity for binding the
modulator protein, thrombomodulin. In the complex with thrombomodulin, thrombin
assumes the slow form and modifies Protein C to APC, activating it. When Protein S, a
vitamin K dependant cofactor, binds APC it enhances its ability to bind to phospholipid
membranes. Then, APC cleaves factors Va and Xa. The deceleration of the production
of thrombin and the clotting cascade is a negative feedback loop.3
Figure 1. Blood Clotting Cascade.4
4
Structure:
The three dimensional structure of thrombin5, along with other serine protease
enzymes, evolved remarkably well to accommodate the chemical transformation that it
catalyzes: amide bond cleavage. The active sites and specificity sites are the most
important components of the enzyme. The motif that has been conserved in serine
proteases is called the catalytic triad, which includes a Ser nucleophile, a His general
acid/base catalyst and a carboxylate, Asp or Glu, assisting with a proton bridge. These
enzymes also have a specificity site called an oxyanion hole, which helps to stabilize the
negative charge on the carbonyl oxygen during the reaction. The catalytic triad is often
numbered as Ser195, His 57, and Asp102, as in trypsin and chymotrypsin.6
Specificity is extremely important in thrombin activity. When referring to
specificty, there is a common nomenclature referring to different residues on the enzyme
and substrate commonly used by enzymologists. The residues on the substrate (P) on
either side of the secile bond are identified as P1 - P1`, by increasing numbers going away
from the sessile bond, Pi` (i = 1, 2, 3…) toward the C terminus and Pi (i = 1, 2, 3…)
toward the N terminus. These correspond to similarly identified residues on the enzyme
labeled with an S. 6;7
S2
S2`
P3 P2 P1 P1` P2` P3`
S3
S1 S1`
Substrate
S3`
For example, in thrombin, Asp189 makes up the S15;8 site and is crucial in the
recognition of sustrates because it forms an ion pair with an Arg or Lys residue which
makes up the P1 site of the substrate. This forms the salt bridge that makes up the S1-P1
5
interaction and helps maintain high specificity. The Leu59-Asn62 and Leu144-Gly150
insertion loops are thought to be key in providing specificity.
There are several other major elements that make up thrombin. They include: the
fibrinogen recognition exosite (anion binding site 1), the heparin binding site (anion
binding site 2), and the RGD sequence. The fibrinogen recognition site is relatively far
from the active site and is involved in binding fibrinogen, thrombin receptor, fibrin, and
thrombomodulin. The heparin binding site is the strongest positively charged patch on thrombin and is involved in the binding of heparin. It is also suggested that this site is
involved in the binding of thrombin to platelets. The RGD sequence is close to the active
site and is said to be the platelet binding site.8 5
Thrombin’s tertiary structure has been studied in great detail and can be defined
by simple descriptions. Moving to the qarternary structure, however, thrombin is more
difficult to define because of its allostery. It has been found that thrombin has a fast and
a slow form, contributing to its role in coagulation and anti-coagulation respectively.9;10
The fast form dominates in high concentrations of Na+ and has a higher specificity for
fibrinogen. The slow form dominates in lower concentrations of Na+ and has a higher
specificity for protein C. Na+ binds upon fibrinogen binding and is released when protein
C binds. The Na+ binding site has been intensely examined and is conserved in many
different species, which proves its importance to the function of the enzyme.11 These
structural elements all contribute to the effectiveness of thrombin.
6
Function:
In thrombin-catalyzed reactions, there are three main steps that enable thrombin to
cleave the peptide bond, substrate binding, acylation and deacylation.6;12 A schematic of
the reaction is shown in Scheme 1 and the elementary steps are shown in Scheme 2.13
Scheme 1.
k1
k2
E + S ES  ES` + P`
k-1
k3
E + P``
In the first step, the enzyme (E) binds the substrate (S), an enzyme-substrate
complex (ES) is formed. The chemical reactions begin with the nucleophilic attack of
Ser195 on the carbonyl carbon of the peptide bond. This attack is promoted by the
transfer of a proton from Ser195 to His 57 and by the stabilizing effect of Asp102 on the
developing positive charge on His57 through the carboxylate group. A tetrahedral
intermediate is produced , which then breaks down when the C-N bond cleaves. This
bond cleavage is promoted by the transfer of the proton from His57 to the leaving group.
The acylation step, represented by k2, produces an acylenzyme (ES`) along with one of
the products (P`), a peptide fragment with a new N terminus. The deacylation step,
represented by k3, releases the enzyme along with a second product (P``), a peptide
fragment with a new C terminus. Through general-base catalysis by His57, the
nucleophilic attack of water on the acyl enzyme causes the hydrolysis of the acyl
fragment from thrombin. Both acylation and deacylation involve a possible proton
bridge and/or two steps of proton transfer. Experiments have shown that this occurs
through proton transfer at the catalytic triad, as in other serine protease enzymes. This
process effectively accelerates the reaction by 17-19 orders of magnitude.14;15
7
8
Catalysis by thrombin, like all serine proteases, obeys Michaelis-Menten kinetics.
If the rates are measured at increasing substrate concentrations, a parabola will be formed
that has an asymptote representing Vmax.
v = Vmax[S]
KM + [S]
(2)
Under steady state conditions, the parameters kcat and KM, the turnover number
and the Michaelis constant, respectively, are given by the following equations.
kcat = k2k3
k2 + k3
;
kcat = Vmax
[Eo]
(3)
KM = Ks
;
Ks = k-1 +k2
k1
(4)
k3__
k2 + k3
When acylation is the limiting step, k3 >> k2, kcat = k2 and KM = Ks. When deacylation is
the limiting step, k2 >> k3, kcat = k3 and KM = Ks k3/ k2.
The bimolecular encounter of the enzyme and substrate is described by a second
order rate constant, kcat / KM. This value is expressed by the following equation.
kcat _ = _____ k1k2k3______
KM
k -1k2 + k -1k3 + k2k3
(5)
When k3 >> k2 or k -1, kcat / KM = k1k2/(k -1+ k2) and when k2 or k3 >> k -1, kcat / KM = k1.
The reaction is first order in [S] and second order over all at low substrate
concentrations, when [S] << KM. The reaction is zero order in [S] and first order overall
at high substrate concentrations, when [S] >> KM, showing saturation of the enzyme.
Under these conditions, kcat measures mostly the actual transformation of the substrate.
9
Small Peptide Anilide Substrates:
Natural substrates of serine protease enzymes are not easily detected analytically
and are usually quite expensive. However, substrate mimics can be used to study the
mode of proton transfer or effect of individual S-P or S’-P’ interactions on the efficiency
of catalysis. Substrate mimics are usually short petide chains with a good leaving group
releasing an easily identifiable signal molecule. The most widely used leaving group for
serine protease studies is a para-nitro-aniline (pNA) for two very practical reasons. First,
the cleavage of the nitroanalide-peptide bond catalyzed by the enzyme strongly resembles
the natural process and pNA is released in proportion to the enzymatic activity. Second,
the absorption maximum of free pNA after cleavage is much different than when it is still
attached to the substrate. Before cleavage, the peptide is colorless with an absoption
maximum in the UV. As pNA is released, it can be easily monitored at its absorption
maximum of 400nm.
The substrates used for these reactions have very small chains that resemble the
natural substrates of the enzyme. These substrates, N-Benzoyl-Phe-Val-Arg-pNA and ND-Phe-Pip-Arg-pNA, are not as selective as the natural substrates, since natural
substrates are much larger and interact with different binding pockets and selectivity
regions of the enzyme. These substrates do, however, provide the S1/P1, S2/P2, and
S3/P3 interactions that occur in natural substrates.
Solvent Isotope Effects and Proton Inventories:16-18
Serine proteases, through the activity of the catalytic triad, function through
general acid-base catalysis that involves proton transfer and proton bridges. One
question that can be posed regarding this mechanism is how active a role do the proton
10
transfers and bridges play in catalysis. In the rate-determining step of the reaction, the
mode of proton interaction can be most accurately measured by solvent isotope effect and
partial solvent isotope effect. The theory behind this method explains that rapidly
exchanging protonic sites will equilibrate with the solvent and in the presence of heavy
water, these protonic sites will be replaced by deuterium. Rate measurements are taken
in buffered water, heavy water, and mixtures of the two. Since the catalytic triad contains
these rapidly exchanging protonic sites, proton transfer to and from these sites will be
replaced by deuterium in the presence of heavy water. Because of its smaller massdependent vibrational frequencies, deuterium reacts slower than protium and, therefore,
catalytic reactions will proceed slower in deuterium oxide than in protium oxide.
Reactions in a mixture of the two will proceed at a rate somewhere between that in water
and heavy water. The rate ratios produced in buffered water and heavy water are highly
dependent on the atom fraction of D, n, in the solution. The equation, derived by Gross
and Bulter, shows the relationship as:
TS
Vn =
Vo i (1-n+nTi)
RS
/j (1-n+nRj)
(6)
Where Vn is velocity in a mixed solvent, Vo is velocity in water, n is atom fraction of
deuterium, RS is reactant state, R is RS fractionation factor, TS is the transition state,
and T is TS fractionation factor. The TS product is over the TS fractional factor and the
RS product is over the RS fractionation factor. In essence, the fractionation factors are
inverse equilibrium isotope effects, KD/KH, for the exchange between a structural site on
RS or TS and a bulk solvent site. Through the least squares fitting procedure, the
contributing isotope effects can be determined. Many complex models can be derived
from this equation, however, the most common simplifications involve the assumption of
11
a unit fractionation factor of most RSs and that, in most hydrolytic enzymes, there are
one or two active-site units that contribute, producing: 1) Vn = Vo(1-n+nT) or 2) Vn =
Vo(1-n+nT1)(1-n+nT2), respectively. In order to acquire a correct proton inventory, the
pH/pD dependence must be known for the reaction. Then, the reactions need to be
performed at a pH plateau in identical H2O and D2O solutions. The models can be
applied to the data and the best fit is based on chemical reasoning and statistical
analysis.16-18
Goal:
The purpose of this study was to identify the number of protonic sites and
characterize their role in catalysis by thrombin in the hydrolysis of chromogenic
substrates that contain some of the P1-P3 specificity sites.
Experimental
Solutions: Tris buffer stock solutions were made with 0.0067M Tris(hydroxymethyl)aminomethane hydrochloride, 0.0133M Tris(hydroxymethyl)-aminomethane, 0.1% PEG,
and 0.03M NaCl for both H2O and D2O. Buffer combinations were made using the two
stock solutions in the following proportions: 15% D2O/85% H2O, 33%D2O/67% H2O,
50% D2O/50% H2O, 67% D2O/33% H2O, 85% D2O/15% H2O.
N-Benzoyl-Phe-Val-Arg-pNA HCl (Sigma-Aldrich) and N-D-Phe-Pip-Arg-pNA
(Diapharma) were dissolved in DMSO for the stock solutions. For Michaelis-Menten
analysis, N-Benzoyl-Phe-Val-Arg-pNA stock solution was used at a concentration of
0.01952M and was diluted progressively from 4KM to 0.04KM. For initial rates
experiments using N-Benzoyl-Phe-Val-Arg-pNA, a 0.01952M stock solution was used
12
and diluted to 3.2 x 10-4 M in the cuvette and for initial rates studies using N-D-Phe-PipArg-pNA, a 3.2 x 10-3 M stock solution was used and diluted to 6.4 x 10-5M in the
cuvette. For progress curve analysis, a 4.88x 10-4 M N-Benzoyl-Phe-Val-Arg-pNA stock
solution was used and diluted to 9.76 x 10-6 M in the cuvette.
Enzyme solutions were made with human alpha thrombin purchased from
Enzyme Research Laboratories, Inc. with activity of 3181 NIH units/mg. These were
diluted with buffer to (5-2) x 10-6 M and, thus were at (5-2) x 10-8 M in the cuvette.
Kinetics: Rate measurements were taken by a Perkin-Elmer Lambda 6 UV/Vis
Spectrophotometer taking 1000 data points at 400nm using a program called PECSS.
The temperature for Michaelis-Menten experiments was regulated by a Techne Tempette
TE-8A circulating water bath and for all of the other experiments, by a Brinkmann MGW
Lauda RM-20 circulating water bath. In all kinetic experiments, 970L buffer, 20L
substrate stock solution, and 10L enzyme stock solution was used.
Michaelis-Menten experiments: The buffer was inserted into the cuvette and incubated in
the cell compartment for 10 minutes. At that point, substrate and enzyme were added and
1000 absorption data points were acquired. This was done with all of the substrate
concentrations and the PECSS software was used to calculate the slope (OD/sec) of each
run. The data was entered into the computer program Grafit and fitted to a MichaelisMenten equation, where Vmax and Vmax/KM were calculated. An example is shown in
Figure 2.
Initial rates: The buffer was incubated in the cell compartment for 5 minutes; then,
enzyme was added and the mixture was incubated for another 10 minutes. Substrate was
13
added and absorption data points were acquired to get the initial rate of the reaction.
PECSS was used to calculate the OD/sec.
8
4
1e 3
V,
OD/sec
6
2

0 

0
2
4


[
S
]
,
m
M

Figure 2. Michaelis-Menten profile for the thrombin-catalyzed hydrolysis of N-BenzoylPhe-Val-Arg-pNA in 0.02M Tris buffer at pH 8.3 containing 0.03M NaCl, 0.1%PEG4000 and at 25  0.2 oC.
Progress curve analysis: These experiments were set up exactly like the initial rate
studies except the reactions were allowed to run to almost completion so the entire curve
could be seen. The data was taken and fit to the first-order rate equation using the
program Grafit. Rate constants were calculated. An example is shown in Figure 3.
14
0 .1 8
0 .1 6
OD
0 .1 4
0 .1 2
0 .1
0 .0 8
0
1
0
0
2
0
0
3
0
0
4
0
0
s
e
c
Figure 3. Progress curve of thrombin-catalyzed hydrolysis of N-Benzoyl-PheVal-Arg-pNA in 0.02M Tris buffer at pH 8.3 containing 0.03M NaCl, 0.1%PEG-4000
and at 37  0.2 oC.
Data Analysis: For each data set, each calculated value was compared to the value found
in the D2O buffer and the rate constant ratios were plotted against the corresponding n
value. For example, for the initial rate data, a vn:vD2O ratio was the average of three
repeats under a set of conditions, calculated and divided by the average value of the rate
of rate constant obtained in D2O, and plotted against n. These curves were fitted to
different proton inventory models where solvent isotope effect and TS/S were
calculated. The model with the lowest reduced chi2 was plotted. The models are shown
in Table 1.
15
Table 1: Models for fitting proton inventory data.
Information obtained
TS1
TS1, solv.
2TS1
2TS1, solv.
TS1, TS2
TS1, TS2, solv.
TS, RS
Equation
Vn = VH (1 - n + n 1)
Vn = VH (1 - n + n 1) Sn
Vn = VH (1 - n + n )2
Vn = VH (1 - n + n )2 Sn
Vn = VH (1 - n + n 1)(1 - n + n 2)
Vn = VH (1 - n + n 1)(1 - n + n 2) Sn
Vn = VH (1 - n + n 1)/(1 - n + n R)
Results:
Table 2 shows examples of fitting data to various models to determine the
fractionation factors for the thrombin-catalyzed hydrolysis of N-Benzoyl-Phe-Val-ArgpNA. The model of choice is give in bold face.
Table 2. Fractionation factors fitting various models for the thrombin-catalyzed
hydrolysis of N-Benzoyl-Phe-Val-Arg-pNA in 0.02M Tris buffer at pH 8.3 containing
0.03M NaCl, 0.1%PEG-4000, and 2% DMSO and at 25.0 0.1oCa or 37  0.2 oCb. Data
were fit using robust and simple weighting.
(vmax)n/(vmax)Da
(kobs)n/(kobs)Db
model
TS1
TS2
Chi^2
TS1
2TS1
TS1, TS2
TS1
0.36  0.07
0.61  0.51
0.25  0.10
0.58  0.09
------------------0.72  0.60
----------
0.056
0.049
0.24
0.029
2TS1
TS1, TS2
0.76  0.06
0.92  0.67
---------0.39  0.11
0.028
0.099
The proton inventory curves for the chosen models of thrombin-catalyzed
hydrolysis of N-Benzoyl-Phe-Val-Arg-pNA are presented in Figure 4 for initial rates, in
Figures 5 and 6 for Michaelis-Menten curves, and in Figure 7 for the progress curves.
The straight line shows the fit for the first model in Table 1 for a single proton bridge at
the transition state.
16
2 .2
2
/v
v
n
D
1 .8
1 .6
1 .4
1 .2
1
0
0
.
2
0
.
4
0
.
6
0
.
8
1
n
/(V
(V
max
)
n
max
)
D
Figure 4. Proton inventory for the thrombin-catalyzed hydrolysis of N-Benzoyl-PheVal-Arg-pNA in 0.02M Tris buffer at pH 8.3 containing 0.03M NaCl, 0.1%PEG-4000,
and 2% DMSO and at 37  0.2 oC.
3 .2
3
2 .8
2 .6
2 .4
2 .2
2
1 .8
1 .6
1 .4
1 .2
1
0
0
.
2
0
.
4
0
.
6
0
.
8
1
n
Figure 5. Proton inventory for the thrombin-catalyzed hydrolysis of N-Benzoyl-PheVal-Arg-pNA in 0.02M Tris buffer at pH 8.3 containing 0.03M NaCl, 0.1%PEG-4000,
and 2% DMSO and at 25  0.1 oC.
17
1 .2
(V
max
/K
M
)
/(V
1 .4
n
max
/K
M
)
D
1 .6
1
0
0
.
2
0
.
4
0
.
6
0
.
8
1
n
Figure 6. Proton inventory for the thrombin-catalyzed hydrolysis of N-Benzoyl-PheVal-Arg-pNA in 0.02M Tris buffer at pH 8.3 containing 0.03M NaCl, 0.1%PEG-4000,
and 2% DMSO and at 25  0.1 oC.
1 .8
D
1 .4
n
k
/k
1 .6
1 .2
1
0 .8
0
0
.
2
0
.
4
0
.
6
0
.
8
1
n
Figure 7. Proton inventory for the thrombin-catalyzed hydrolysis of N-Benzoyl-PheVal-Arg-pNA in 0.02M Tris buffer at pH 8.3 containing 0.03M NaCl, 0.1%PEG-4000,
and 2% DMSO and at 37  0.2 oC. Pseudo first-order kinetics.
18
Figure 8 shows a proton inventory curve for the hydrolysis of N-D-Phe-Pip-ArgpNA studied by the initial rate method.
1
/v
v
n
D
0 .8
0 .6
0 .4
0
0
.
2
0
.
4
0
.
6
0
.
8
1
n
Figure 8. Proton inventory for the thrombin-catalyzed hydrolysis of N-D-Phe-Pip-ArgpNA in 0.02M Tris buffer at pH 8.3 containing 0.3M NaCl, 0.1%PEG-4000, and 2%
DMSO and at 37  0.2 oC.
Table 3 has the compilation of solvent isotope effects and fractionation factors for
the thrombin-catalyzed hydrolysis of N-Benzoyl-Phe-Val-Arg-pNA.
Table 3. Summary of solvent isotope effects and fractionation factors from all
calculations of the thrombin-catalyzed hydrolysis of N-Benzoyl-Phe-Val-Arg-pNA in
0.02M Tris buffer at pH 8.3 containing 0.03M NaCl, 0.1%PEG-4000 and at a25.0 ± 0.1
°C or b37 ± 0.2 °C.
SIE
TS/
S
Vmaxa
Vmax/KMa
kobsb
Vb
2.8 0.2
1.2 0.2
1.6 0.1
2.21 0.03
0.37 0.07 0.3 0.3; 2.3 1.8 0.58 0.09 0.39 0.04; 1.2 0.1
19
The solvent isotope effect for Vmax for the thrombin-catalyzed hydrolysis of N-DPhe-Pip-Arg-pNA was 2.8 ± 0.1 and the best model gave a TS of 0.4 ± 0.05. The
solvent isotope effect for Vmax/KM was 1.0 ± 0.4.
Discussion:
The reactions that were the target of the proton inventory studies were the human
alpha thrombin-catalyzed hydrolysis of two chromogenic anilides, Bz-Phe-Val-Arg-pNA
and H-D-Phe-L-Pip-Arg-pNA. These substrates contained sequences that were optimal
for the P1- P3 sites. The optimal pH was found to be between pH 8 and 8.5 in Tris
buffer. Bz-Phe-Val-Arg-pNA was found to have limited solubility under experimental
conditions at approximately 3 x 10-4 M (4KM). The salt level was kept below optimal at
0.03 M NaCl in order to optimize enzyme saturation. For the hydrolysis by thrombin of
fibrinogen and its short substrate mimics, the NaCl concentration in buffer is kept at 0.3
M. Classic Michaelis-Menten kinetics was observed in all cases.
Solvent isotope effects for the thrombin-catalyzed hydrolysis of the two tripeptide
pNA substrates were 2.8 – 3.15 for Vmax and between 1 and 1.6 for Vmax/KM, and similar
to cases previously studied. Proton inventory or partial solvent isotope effect studies
permitted resolving the solvent isotope effects into their contributing components: proton
bridges occurring at the TS of the rate-determining step in thrombin catalysis and other
contributions from solvating hydrogen bonds. The preferred model for isotope sensitive
sites was determined in each case through two steps. The first step was choosing the data
with the lowest reduced chi2. The second step involved looking at all of the models and
making sure there was consistency between data at low substrate concentration and
saturation.
20
From the data, it can be concluded that at low substrate concentrations, diffusion
may play a large role in the reaction and at high substrate concentrations, diffusion does
not contribute to the reaction. The Vmax/KM data has a very large error, but the small
value of the solvent isotope effect and previous experiences support the suggestion that
the reaction at low substrate concentrations is partly limited by the rate of diffusion. The
data from the initial rate experiments show a small solvation factor even though the
experiment is done at high substrate concentrations, but it is to be expected. The v values
are not extrapolated to Vmax, so there is a slight contribution from solvation and a slightly
lower solvent isotope effect, both of which can be seen in Figure 4. Figure 5 further
proves this trend. The proton inventory in Vmax shows no contribution of solvation to the
solvent isotope effect. This is consistent with rate-determining formation of the C-O
bond or breakdown of the C-N bond on Scheme 2.
Enyedy and Kovach studied the thrombin catalyzed hydrolysis of fluorogenic
substrates Z-Pro-Arg-7-amido-4-methylcoumarin (7-AMC), N-t-Boc-Val-Pro-Arg-7AMC, and 2) internally quenched fluorogenic peptides a) (AB)Val-Phe-Pro-Arg-SerPhe-Arg-Leu-Lys(DNP)-Asp-OH, the optimal substrate; b) (AB)Val-Ser-Pro-Arg-SerPhe-Gln-Lys(DNP)-Asp-OH, recognition sequence for factor VIII. With simple
fluorogenic substrates, the KSIEs for kcat are near 3 and for two intra-molecularly
fluorescence-quenched substrates, they are 2.2 ± 0.2. The fractionation factors for kcat and
kcat/Km for the rate-determining chemical step in acylation of the enzymes by the
substrates studied are nearly identical. In thrombin-catalyzed reactions, one or two
protons participate in the rate-determining catalytic process with intrinsic KSIEs between
2 and 2.8.
21
The fractionation factors are near 1.0 ± 0.4 for solvation terms at saturating
concentrations of the substrates. The KIEs for the kcat/Km term are between 1 and 2.2.
They all present curved proton inventories and include a contribution from solvation.
Two identical proton transfer sites seem to be involved in catalysis of the hydrolysis of
substrates containing certain P1-P3 residues. The presence of P1’-P5‘residues in the
substrates of thrombin elicit large solvent rearrangements even in the kcat term in one
case. This is consistent with the recognized conformational change in thrombin during
the activation of fibrinogen.
The proton inventory studies of reactions catalyzed by blood clotting enzymes
had not been performed previously except the thrombin-catalyzed hydrolysis of the
minimum substrate Z-Arg-ethyl ester.19 Those experiments showed the reaction to
involve one-proton catalysis. Lottenberg et al.20 suggested that the unique pH
dependence of the hydrolysis of a series of oligopeptide substrates was the result of two
or three protons participating in the mechanism. KSIEs of >3 were reported by Stone et
al.15 which lent support to the anticipation of multi-proton catalysis by thrombin if the
requirements for optimal interactions between enzyme and substrate subsites were
available. Conclusions from the present study agree fully with these earlier results and
predictions.
22
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