Mechanistic Studies on Enzymatic Reactions by Electrospray Ionization MS Using a Capillary Mixer with Adjustable Reaction Chamber Volume for Time-Resolved Measurements

Article abstract of DOI:10.1021/ac0355348

Mass spectrometry (MS)-based techniques have enormous potential for kinetic studies on enzyme-catalyzed processes. In particular, the use of electrospray ionization (ESI) MS for steady-state measurements is well established. However, there are very few reports of MS-based studies in the pre-steady-state regime, because it is difficult to achieve the time resolution required for this type of experiment. We have recently developed a capillary mixer with adjustable reaction chamber volume for kinetic studies by ESI-MS with millisecond time resolution (Wilson, D. J.; Konermann, L. Anal. Chem. 2003, 75, 6408-6414). Data can be acquired in kinetic mode, where the concentrations of selected reactive species are monitored as a function of time, or in spectral mode, where entire mass spectra are obtained for selected reaction times. Here, we describe the application of this technique to study the kinetics of enzyme reactions. The hydrolysis of p-nitrophenyl acetate by chymotrypsin was chosen as a simple chromophoric model system. On-line addition of a "makeup solvent" immediately prior to ionization allowed the pre-steady-state accumulation of acetylated chymotrypsin to be monitored. The rate constant for acetylation, as well as the dissociation constant of the enzyme-substrate complex obtained from these data, is in excellent agreement with results obtained by conventional stopped-flow methods. Bradykinin was chosen to illustrate the performance of the ESI-MS-based method with a nonchromophoric substrate. In this case, the unfavorable rate constant ratio for acylation and deacylation of the enzyme precluded measurements in the pre-steady-state regime. Steady-state experiments were carried out to determine the turnover number and the Michaelis constant for bradykinin. The methodologies used in this work open a wide range of possibilities for future ESI-MS-based kinetic assays in enzymology.

Full text of DOI:10.1021/ac0355348

Anal. Chem. 2004, 76, 2537-2543
Me c h a n is t ic S t u d ie s o n En zym a t ic Re a c t io n s b y
Ele c t ro s p ra y Io n iza t io n MS Us in g a Ca p illa ry Mix e r
w it h Ad ju s t a b le Re a c t io n Ch a m b e r Vo lu m e fo r
Tim e -Re s o lve d Me a s u re m e n t s
De re k J . Wils on a nd La rs Kone rm a nn*
Department of Chemistry, The University of Western Ontario, London, ON, N6A 5B7, Canada
most important tools for elucidating these reaction mechanisms.
Immediately after the initiation of an enzymatic reaction, there is
a short period of time (milliseconds to seconds, depending on
the rate constants involved) during which reaction intermediates
become successively populated. It is during this “pre-steady-state”
period that the rate constants of individual steps can be measured.
It is often possible to directly deduce reaction mechanisms based
on pre-steady-state studies, whereas this is usually not the case
for the more commonly employed steady-state measurements.2-4
With very few exceptions,5 pre-steady-state kinetic studies
require a time resolution in the millisecond range, which can only
be achieved by using automated rapid mixing techniques. Stopped-
flow rapid mixing involves quickly flushing reactants through a
mixer and into an observation cell. The flow is then stopped, and
the reaction is monitored in real time by optical methods.6
Quench-flow experiments also involve rapid mixing, but the
reaction is quenched after predetermined delay times through
addition of a suitable agent (e.g., acid, base, or organic solvent).
Subsequently, the mixture is analyzed off-line, e.g., by chroma-
tography-based methods.7 For continuous-flow studies, reactants
are continuously passed through a mixer and into a reaction
capillary. The reaction time at any point along this capillary
depends on the tube dimensions and on the flow rate used.
Continuous-flow methods can have a time resolution in the
submillisecond range.8
Mass spectrometry (MS)-based techniques have enor-
mous potential for kinetic studies on enzyme-catalyzed
processes. In particular, the use of electrospray ionization
(ESI) MS for steady-state measurements is well estab-
lished. However, there are very few reports of MS-based
studies in the pre-steady-state regime, because it is
difficult to achieve the time resolution required for this
type of experiment. We have recently developed a capillary
mixer with adjustable reaction chamber volume for kinetic
studies by ESI-MS with millisecond time resolution (Wil-
son, D. J.; Konermann, L. An a l. Ch em . 2003, 75, 6408-
6 4 1 4 ). Data can be acquired in kinetic mode, where the
concentrations of selected reactive species are monitored
as a function of time, or in spectral mode, where entire
mass spectra are obtained for selected reaction times.
Here, we describe the application of this technique to
study the kinetics of enzyme reactions. The hydrolysis of
p-nitrophenyl acetate by chymotrypsin was chosen as a
simple chromophoric model system. On-line addition of
a “makeup solvent” immediately prior to ionization al-
lowed the pre-steady-state accumulation of acetylated
chymotrypsin to be monitored. The rate constant for
acetylation, as well as the dissociation constant of the
enzyme-substrate complex obtained from these data, is
in excellent agreement with results obtained by conven-
tional stopped-flow methods. Bradykinin was chosen to
illustrate the performance of the ESI-MS-based method
with a nonchromophoric substrate. In this case, the
unfavorable rate constant ratio for acylation and deacyla-
tion of the enzyme precluded measurements in the pre-
steady-state regime. Steady-state experiments were car-
ried out to determine the turnover number and the
Michaelis constant for bradykinin. The methodologies
used in this work open a wide range of possibilities for
future ESI-MS-based kinetic assays in enzymology.
Typically, the kinetics in these different types of rapid mixing
experiments are monitored optically, e.g., by UV-visible absorp-
tion or by fluorescence spectroscopy. However, most reactions
of enzymes with their “natural” substrates cannot be studied in
this way because there are no associated chromophoric changes.
For this reason, kineticists often use artificial substrate analogues
that undergo a color change upon turnover. Obviously, this
approach is problematic because the kinetics observed with these
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Biochemistry 1 9 9 9 , 38, 6537-6546.
Enzyme catalysis is a vital component of all biological systems.
Enzyme mechanisms range from simple two-step processes to
complex multistep reactions.1 Kinetic experiments are among the
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* Corresponding author: (e-mail) konerman@uwo.ca; (phone) (519) 661-2111
ext. 86313; (fax) (519) 661-3022; http:/ / publish.uwo.ca/ ∼konerman.
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10.1021/ac0355348 CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/01/2004
Analytical Chemistry, Vol. 76, No. 9, May 1, 2004 2537

analogues are often different from those that would be observed
with the natural substrate(s).9 In some cases, the use of radioac-
tively labeled substrates provides an alternative approach.10,11
However, radiochemical methods are somewhat cumbersome, and
problems can arise due to nonspecific entrapment of the label.
In recent years, mass spectrometry (MS)-based techniques
have shown great promise in the area of chemical and biochemical
kinetics.12-21 The most significant advantage offered by MS-based
studies is that they do not require chromophoric substrates or
radioactive labeling. Consequently, there is great interest in the
application MS for kinetic studies on enzyme-catalyzed pro-
cesses.9,22-27 Electrospray ionization mass spectrometry (ESI-MS),
in particular, has enormous potential as an alternative to the
traditional methods for monitoring enzyme kinetics, because the
reaction mixture can often be injected directly into the ion source
for on-line analysis, while the reaction occurs in solution. This
approach allows the identification of reactive species based on
their mass-to-charge ratio, their MS/ MS characteristics, or both,
while an analysis of the measured intensity-time profiles can
provide reaction rates.28 Our group has recently developed a
capillary mixer with adjustable reaction chamber volume for on-
line kinetic studies by ESI-MS.29 This system allows the monitor-
ing of processes having rate constants in the range from ∼1 s-1
up to at least 100 s-1. When used in conjunction with a quadrupole
mass spectrometer, reaction kinetics can be monitored either by
measuring entire mass spectra for selected reaction times (“spec-
tral mode”) or by recording detailed intensity-time profiles for
selected ions (“kinetic mode”). This unique combination of
features should make this device a powerful tool for on-line studies
on enzyme-catalyzed processes, particularly in the pre-steady-state
regime.
Chymotrypsin is a member of the serine protease family.30-32
Ser195 represents the reactive nucleophile in the active site of this
enzyme. Although the physiological role of chymotrypsin is to
serve as an endopeptidase, it also catalyzes the hydrolysis of
esters, including numerous synthetic substrate analogues. Chy-
motrypsin shows a moderate degree of specificity for aromatic
or bulky aliphatic substrates; hydrolytic cleavage occurs prefer-
entially at the C-terminal side of phenylalanine, tyrosine, tryp-
tophan, or leucine.33 The generally accepted reaction mechanism
for chymotrypsin-catalyzed hydrolysis is depicted in Scheme 1.1-3
S c h e m e 1
In the first step of this reaction sequence, the enzyme E and
the substrate S form a noncovalent enzyme-substrate complex,
ES, that is characterized by the dissociation constant Kd. Subse-
quently, Ser195 forms a covalent bond with the carbonyl carbon of
the substrate, thus releasing the first hydrolysis product P1. The
rate constant of this acylation step is denoted as k2. The
subsequent deacylation has a rate constant of k3, and it leads to
regeneration of the free enzyme by hydrolysis of the Ser195-ester
bond, through release of the second hydrolysis product P2. For
conditions where S is present in large excess, it can be shown
that the concentration of P1 as a function of time t in the pre-
steady-state regime is given by2,3
[P1](t) ) C1(1 - exp(-kobst)) + C2t
(2)
and the concentration-time profile of the covalent EP2 complex
can be expressed as
(9) Northrop, D. B.; Simpson, F. B. Bioorg. Med. Chem. 1 9 9 7 , 5, 641-644.
(10) Anderson, K. S.; Sikorski, J. A.; Johnson, K. A. Biochemistry 1988, 27, 7395-
7406.
[EP2](t) ) C3(1 - exp(-kobst))
(3)
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(12) Miranker, A.; Robinson, C. V.; Radford, S. E.; Aplin, R.; Dobson, C. M. Science
1 9 9 3 , 262, 896-900.
(13) Sam, J. W.; Tang, X. J.; Magliozzo, R. S.; Peisach, J. J. Am. Chem. Soc. 1 9 9 5 ,
Consequently, the sum of the concentrations of free enzyme and
ES complex are given by
117, 1012-1018.
(14) Yang, H.; Smith, D. L. Biochemistry 1 9 9 7 , 36, 14992-14999.
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292.
([Efree] + [ES])(t) ) C4 exp(-kobst) + C5
(4)
(17) Gross, J. W.; Hegemann, A. D.; Vestling, M. M.; Frey, P. A. Biochemistry
2 0 0 0 , 39, 13633-13640.
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5905.
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4630.
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Spectrom. 2 0 0 0 , 14, 772-776.
C1, ..., C5 in these expressions are constants and kobs is given by
k2[S]
kobs ) k3 +
(5)
Kd + [S]
(21) Kolakowski, B. M.; Konermann, L. Anal. Biochem. 2 0 0 1 , 292, 107-114.
(22) Northrop, D. B.; Simpson, F. B. FASEB J. 1 9 9 7 , 11, A1021.
(23) Paiva, A. A.; Tilton, R. F.; Crooks, G. P.; Huang, L. Q.; Anderson, K. S.
Biochemistry 1 9 9 7 , 36, 15472-15476.
where [S] is the substrate concentration. Measurements of kobs
as a function of substrate concentration allow the determination
of the parameters k2, k3, and Kd in Scheme 1.
(24) Zechel, D. L.; Konermann, L.; Withers, S. G.; Douglas, D. J. Biochemistry
1 9 9 8 , 37, 7664-7669.
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2 0 0 0 , 72, 3311-3319.
For t . 1/ kobs, the exponential terms in eqs 2-4 become
negligible, thus marking the transition from the pre-steady-state
to the steady-state regime. Under steady-state conditions, [EP2],
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40, 3774-3779.
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Am. Chem. Soc. 2 0 0 3 , 125, 9938-9939.
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8790.
111, 4600-4604.
(29) Wilson, D. J.; Konermann, L. Anal. Chem. 2 0 0 3 , 75, 6408-6414.
(33) Hess, G. P. Enzymes 1 9 7 1 , 3, 213-248.
2538 Analytical Chemistry, Vol. 76, No. 9, May 1, 2004

[Efree], and [ES] remain constant, whereas [P1] and [P2] increase
linearly with time. The rate of reaction under these steady-state
conditions is given by the Michealis-Menten expression1
compared to our earlier setup is the addition of a mixing “tee” at
the end of the outer capillary, which allows the addition of an
“ESI-friendly” makeup solvent to the reaction mixture, immediately
prior to ionization. The makeup solvent was infused at a flow rate
of 40 µL/ min, for a total flow rate of 80 µL/ min at the ESI source.
The second mixer is made from Flexon HP tubing (Alltech,
Deerfield, IL); its two inlets accommodate the end of the outer
capillary and a fused-silica capillary (100 ( 1.5 µm i.d., 167 ( 3
µm o.d., Polymicro Technologies, Phoenix, AZ) for addition of
the makeup solvent. The mixer outlet is connected to a 1-cm fused-
silica capillary (75 ( 1.2 µm i.d., 150 ( 2 µm o.d. Polymicro
Technologies) that ends at the ESI source. Ionization takes place
by pneumatically assisted ESI in the positive ion mode at a sprayer
voltage of 6 kV. All measurements were carried out on a triple
quadrupole mass spectrometer (PE Sciex, API 365, Concord, ON,
Canada). It is noted that the makeup solvents used (see below)
also act as chemical quenchers of the enzymatic reactions studied
here. Therefore, the residence time of the solution in the flow
system downstream of the second mixer (∼30 ms) does not
contribute to the total dead time of the kinetic measurements,
which is also estimated to be ∼30 ms. Analysis of the kinetic data
obtained was carried out based on a framework described
previously that takes into account laminar flow effects in the
reaction capillary.29 Safety considerations pertinent to the operation
of the ESI-MS-coupled capillary mixer have also been discussed
in that previous work. Protein mass spectra were deconvoluted
using the Biomultiview analysis package provided by the instru-
ment manufacturer.
d[P1] d[P2] kcat[E]0[S]
)
)
(6)
dt
dt
KM + [S]
where [E]0 is the total enzyme concentration. Measurements of
the reaction rate as a function of [S], therefore, provide the
turnover number kcat and the Michaelis constant KM.4,34,35
This work explores the application of our recently developed
capillary mixer29 for kinetic studies on enzymatic reactions by ESI-
MS. Using chymotrypsin as a model system, we will initially
describe results obtained with the chromophoric substrate p-
nitrophenyl acetate (p-NPA). The hydrolysis kinetics measured
for this compound by ESI-MS are compared to optical data
obtained by standard optical stopped-flow spectroscopy. Subse-
quently, the ESI-MS-based approach is used for studies on the
hydrolysis of the peptide bradykinin, which represents a non-
chromophoric substrate. It will be seen that the method employed
here can provide detailed information on the kinetics and mech-
anisms of enzyme-catalyzed processes.
EXPERIMENTAL SECTION
Chemicals. Chymotrypsin (nominally in its R form, see below)
and p-NPA were obtained from Sigma (St. Louis, MO). Distilled
grade methanol and hydrochloric acid were supplied by Caledon
(Georgetown, ON, Canada), glacial acetic acid was supplied by
BDH (Toronto, ON, Canada), and ammonium hydroxide was
supplied by Fisher (Nepean, ON, Canada). These chemicals were
used without further purification. Bradykinin, supplied by Bachem
(Torrence, CA), was extensively dialyzed against distilled water
using a 100 MWCO Float-A-Lyzer (Spectrum Laboratories, Rancho
Dominguez, CA) prior to use.
Optical Stopped-Flow Experiments. Optical control experi-
ments were carried out on an SFM-4 instrument (Bio-Logic, Claix,
France) operated in absorbance mode, using an observation
wavelength of 405 nm to monitor the release of p-nitrophenol (p-
NP).36 The two stepper motor-driven syringes were advanced at
3.5 mL s-1 each, for an instrument dead time of 3.3 ms.
Enzymatic Reactions. The limited solubility of p-NPA in
purely aqueous solutions necessitated the use of 20% (v/ v)
methanol in the reaction mixture. Solutions of similar (or even
higher) organic content were used in previous studies on the
chymotrypsin-catalyzed conversion of p-NPA.37,38 The activity of
chymotrypsin does not seem to be affected by the presence of
organic cosolvents at these concentrations.39 Solutions containing
40% methanol and 1-10 mM p-NPA were brought to pH 8.1 using
ammonium hydroxide. These solutions were mixed in a 1:1 ratio
with 32 µM chymotrypsin in water for a final pH of 7.8, which
corresponds to the pH optimum of the enzyme. A makeup solvent
consisting of 5 mM HCl was found to produce the best signal-to-
noise ratio for these p-NPA studies. Experiments on bradykinin
were carried out in an analogous manner, but in purely aqueous
solution, and by using 20% (v/ v) acetic acid in water as makeup
solvent. Control experiments showed the pH of the solutions to
be stable for at least 5 s after mixing. Substrate concentrations
given below represent the values in the reaction mixture, i.e., after
the first mixing step. Burst phase kinetics observed by stopped-
On-Line Kinetic ESI-MS Experiments. ESI-MS-based ki-
netic experiments were carried out on a custom-built continuous-
flow mixing apparatus described previously,29 which has been
coupled to a triple quadrupole mass spectrometer (model Sciex
API 365, Concord, ON, Canada). Briefly, this flow setup consists
of two concentric capillaries that are connected to sample injection
syringes. Reactions are initiated by mixing of two solutions at the
outlet of the inner capillary. The reaction time is determined by
the solution flow rate and by the distance between the mixing
point and the end of the outer capillary. For experiments in kinetic
mode, the inner capillary is steadily withdrawn from the end of
the outer capillary, while the mass spectrometer is set to monitor
selected m/ z values, corresponding to specific solution-phase
species, as a function of time. In spectral mode, the inner capillary
is set at specific distances from the end of the outer capillary,
such that entire mass spectra can be obtained for selected reaction
times.
For the experiments described here, both reactant solutions
were introduced into the apparatus at 20 µL/ min using syringe
pumps (Harvard Apparatus, Saint Laurent, PQ Canada) for a total
flow rate of 40 µL/ min after the mixer. One important modification
(36) Head, M. B.; Mistry, K. S.; Ridings, B. J.; Smith, C. A.; Parker, M. J. J. Chem.
Educ. 1 9 9 5 , 72, 184-186.
(37) Gutfreund, H.; Sturtevant, J. M. Biochem. J. 1 9 5 6 , 63, 656-661.
(38) Anderson, J.; Byrne, T.; Woelfel, K. J.; Meany, J. E.; Spyridis, G. T.; Pocker,
Y. J. Chem. Educ. 1 9 9 4 , 71, 715-718.
(34) Fierke, C. A.; Hammes, G. G. Methods Enzymol. 1 9 9 5 , 249, 3-37.
(35) Johnson, K. A. In The Enzymes, 3rd ed.; Sigman, D. S., Ed.; Academic
Press: San Diego, 1992; Vol. XX, pp 1-61.
(39) Faller, L.; Sturtevant, J. M. J. Biol. Chem. 1 9 6 6 , 241, 4825-4834.
Analytical Chemistry, Vol. 76, No. 9, May 1, 2004 2539

flow UV-visible spectroscopy for p-NPA hydrolysis showed that
the chymotrypsin used had an active enzyme content of 80 wt %.
This factor was taken into account for calculations involving
enzyme concentrations. All experiments were carried out at room
temperature (22 ( 1 °C). pH measurements were performed with
an AB15 pH meter (Fisher Scientific). Reported pH values are
“as read”; i.e., no corrections were made for solutions that
contained methanol.
RESULTS AND DISCUSSION
Forms of Chymotrypsin in Commercially Supplied Sam-
ples. R-Chymotrypsin consists of three polypeptide chains that
are covalently linked by disulfide bridges. The enzyme is gener-
ated from chymotrypsinogen, which is an inactive precursor with
a continuous polypeptide backbone.1,2,40 The formation of R-chy-
motrypsin from chymotrypsinogen can proceed by at least two
mechanisms and involves multiple intermediate species.41 In one
of these scenarios, chymotrypsinogen is first converted to an active
enzyme through cleavage of the Arg15-Ile16 bond. Subsequent
hydrolysis of the Ile13-Ser14 bond releases a dipeptide. The
resulting enzyme is known as δ-chymotrypsin. The R form of the
enzyme (MW 25 234) is formed through two additional hydrolysis
steps that release a second dipeptide consisting of Thr147 and
Asn148. Incomplete removal of the second dipeptide, by hydrolysis
of just one of the peptide bonds, yields a form that can be referred
to as δ′ (MW 25 449).41,42
The deconvoluted ESI mass spectra of the chymotrypsin
samples used for this work show two major components (Figure
1A). The observed peaks at 25 234 ( 1 and 25 450 ( 1 Da
correspond to the masses of R- and δ′-chymotrypsin, respectively.
The intensity ratios of the two species were found to be highly
variable in the different lots used for the current study (compare,
e.g., Figures 1A and 4). These observations are in line with
previous work by Ashton et al.43
Hydrolysis Kinetics of p-NP A. The chymotrypsin-catalyzed
hydrolysis of p-NPA generates p-NP and acetate. In the framework
of Scheme 1, p-NP corresponds to P1 and acetate corresponds to
P2.1-3,33,39 p-NPA was chosen as substrate for these studies,
because the released p-NP is an intense yellow, thus providing a
convenient way to compare the ESI-MS-based kinetic experiments
with the results of optical control experiments.36,38 ESI mass
spectra were generated at various times after mixing the enzyme
solution with p-NPA. Figure 1 shows deconvoluted mass distribu-
tions, obtained at a p-NPA concentration of 2 mM, for three
different reaction times. The two major peaks observed at t ≈ 30
ms (Figure 1A) are assigned to the R and δ′ forms of chymo-
trypsin. For a reaction time of 700 ms (Figure 1B), both forms of
the protein show pronounced satellite peaks that correspond to a
mass increase of 43 Da. At t ) 3 s, these satellite peaks have
become the dominant features in the mass distribution (Figure
1C). The observed mass increase of 43 Da is attributed to the
acetylation of Ser195 in the active site of the enzymes. The three
spectra depicted in Figure 1, therefore, represent the pre-steady-
Fig u re 1 . Deconvoluted ESI mass distributions recorded during the
pre-steady-state of p-NPA hydrolysis by chymotrypsin. Peaks labeled
with R and δ′ correspond to R- and δ′-chymotrypsin, respectively. R-Ac
and δ′-Ac refer to acetylated forms of the two enzyme species,
corresponding to the covalent EP2 complex in Scheme 1. Spectra
were recorded at reaction times of 30 ms (A), 700 ms (B), and 3 s
(C). The p-NPA concentration was 2 mM. All four forms of the protein
(R, R-Ac, δ′, δ′-Ac) show minor satellite peaks corresponding to
masses that are 98 ( 2 Da higher than those of the corresponding
proteins (labeled with /). For R- and δ′-chymotrypsin, these peaks
are also observed in the absence of substrate (data not shown). We
tentatively assign these satellite peaks to adducts of the protein with
an unidentified low molecular weight contaminant. Adduct formation
is a common occurrence in ESI-MS.24,52 As expected,53 the magnitude
of the satellite peaks depends on the declustering voltage in the ion
sampling interface of the mass spectrometer (data not shown).
state accumulation of the EP2 complex in Scheme 1. The fact that
both forms of the protein undergo acetylation confirms that both
of them are catalytically active, as previously observed by Ashton
et al.43 Figure 2 shows pre-steady-state intensity-time profiles of
the unmodified and the acetylated forms of R-chymotrypsin. As
predicted by eq 5, the acetylation rate depends on the substrate
concentration. Consequently, the measured kinetics are markedly
slower at 1 mM p-NPA (Figure 2A) than at 5 mM p-NPA (Figure
2B). Very similar kinetics were observed for δ′-chymotrypsin (data
not shown).
Exponential fits to the measured intensity-time profile provide
the parameter kobs (see eqs 3 and 4). Plots of these kobs values as
a function of p-NPA concentration are depicted in Figure 3 for
both forms of the enzyme. The values measured for δ′-chymo-
trypsin are slightly higher than those for R-chymotrypsin. How-
ever, the differences are small, and the error bars overlap for most
data points. These observations are consistent with previous
studies on chymotrypsin that suggest that the various forms
(40) Freer, S. T.; Kraut, J.; Robertus, J. D.; Wright, H. T.; Xuong, N. H.
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Welham, K. J. FEBS Lett. 1 9 9 1 , 292, 201-204.
2540 Analytical Chemistry, Vol. 76, No. 9, May 1, 2004

accurate determination by this method. This is entirely consistent
with the accepted mechanism of p-NPA hydrolysis by chymo-
trypsin, according to which k3 corresponds to the rate-determining
step in Scheme 1. Previous work has shown k3 to be orders of
magnitude smaller than k2.1-3 This difference in rate constants is
responsible for the fact that the EP2 complex accumulates during
p-NPA hydrolysis, which is a prerequisite for meaningful pre-
steady-state measurements. For this scenario (k3 , k2), the rate
constant k3 can be approximated by kcat. Based on optical steady-
state measurements, we found k3 ≈ kcat to be 0.034 ( 0.003 s-1
(data not shown).
Measurements of kobs as a function of substrate concentration
were also carried out by stopped-flow spectroscopy, using the
release of the yellow p-NP moiety as optical probe. In contrast to
the ESI-MS experiments, these optical studies cannot discern the
two forms of the enzyme, and therefore, the measured data
represent a weighted average of the substrate conversion caused
by R- and δ′-chymotrypsin. The analysis of the optical kinetics
was carried out based on eq 2 (data not shown), and the results
obtained are included in Figure 3, yielding k2 and Kd values of
3.6 ( 0.2 s-1 and 1.6 ( 0.1 mM, respectively. These results are in
good agreement with those reported above, thus confirming the
reliability of our ESI-MS-based method as a tool for monitoring
the kinetics of enzymatic reactions.
Fig u re 2 . Pre-steady-state hydrolysis of p-NPA by chymotrypsin
monitored by ESI-MS in kinetic mode. The two panels depict the
depletion of unmodified R-chymotrypsin (R) and the formation of the
acetylated R-chymotrypsin (R-Ac) at p-NPA concentrations of 1 (A)
and 5 mM (B). The data were obtained by monitoring the 12+ charge
state of free and acetylated enzyme at m/z 2103 and 2107,
respectively. Solid lines are fits to the experimental data based on
eqs 3 and 4.
The k2 values obtained in the different experiments described
here are close to the corresponding rate constant of 3 s-1 that
was previously reported by Gutfreund and Sturtevant.37 Also, our
estimate of k3 is in line with their reported value of 0.03 s-1
.
However, the Kd measurements in that work resulted in a value
of 7 mM, which is substantially higher than the results obtained
here. This discrepancy is not entirely unexpected, however,
considering the much higher salt concentrations used by those
authors, together with the known dependence of Kd on ionic
strength.39
In summary, the pre-steady-state data on the hydrolysis of
p-NPA clearly establish the viability of our ESI-MS-based method
for mechanistic and kinetic studies on enzymatic processes. In
the described experiments, the use of a chromophoric substrate
allowed the independent confirmation of the measured kinetics
by optical stopped-flow spectroscopy. We will now examine the
conversion of a nonchromophoric compound, bradykinin, that
cannot be followed by standard optical methods.
Fig u re 3 . Measured kobs values for p-NPA hydrolysis by chymo-
trypsin as a function of substrate concentration. Solid triangles
represent ESI-MS measurements for R-chymotrypsin, and solid circles
depict the corresponding data for δ′-chymotrypsin. Each point
represents the average of four fits (two intensity-time profiles for the
formation of the acetylated enzymes, and two traces for the depletion
on the nonacetylated forms). Open circles depict kobs values deter-
mined by optical stopped-flow spectroscopy in triplicate measure-
ments. Error bars indicate standard deviations. Fits to these kobs
values based on eq 5 are given as solid line for the δ-chymotrypsin
ESI-MS kinetics, as dashed line for the optical data, and as dash-
dotted line for the R-chymotrypsin ESI-MS kinetics.
Hydrolysis Kinetics of Bradykinin. Bradykinin is a peptide
consisting of nine amino acids (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-
Arg, MW 1060). Based on the known preference of chymotrypsin
to induce hydrolysis on the C-terminal side of phenylalanine,33
both Phe5-Ser6 and Phe8-Arg9 represent potential cleavage sites.
Preliminary studies showed the second of these possibilities to
be preferred by a ratio of at least 100:1 (data not shown). Thus,
P1 in Scheme 1 corresponds to Arg9, whereas P2 is represented
by the remainder of the peptide, i.e., Arg-Pro-Pro-Gly-Phe-Ser-Pro-
Phe (MW 904).
generated during processing of the enzyme have very similar
structures and reaction kinetics.1,40,44
Figure 4 shows the deconvoluted ESI mass distribution of
chymotrypsin, 0.2 s after mixing with 2 mM bradykinin. The
spectrum shows peaks corresponding to R- and δ′-chymotrypsin,
the latter being the dominant species in the enzyme lot that was
used for these bradykinin experiments. In contrast to the kinetic
measurements performed on p-NPA, neither the R nor the δ′ form
Fits to the measured kobs data based on eq 5 yield k2 values of
3.2 ( 0.3 and 3.7 ( 0.3 s-1 for R- and δ′-chymotrypsin, respectively.
The corresponding dissociation constants Kd are 1.4 ( 0.2 and
1.7 ( 0.2 mM. Unfortunately, the value of k3 is too small for an
(44) Niemann, C. Science 1 9 6 4 , 143, 1287-1296.
Analytical Chemistry, Vol. 76, No. 9, May 1, 2004 2541

Fig u re 4 . Deconvoluted ESI mass distribution obtained 0.2 s after
mixing chymotrypsin with 2 mM bradykinin. R and δ′ represent the
two forms of the enzyme, and / indicates adduct peaks, as in Figure
1. Arrows indicate the masses were the EP2 complexes (Arg-Pro-
Pro-Gly-Phe-Ser-Pro-Phe-[Ser195-enzyme]) of R- (26 120 Da) and δ′-
chymotrypsin (26 335 Da) would be expected.
Fig u re 5 . Chymotrypsin-catalyzed hydrolysis kinetics of bradykinin
at three substrate concentrations. The signal intensity of the
[M + H]+ ion, corresponding to the hydrolysis product P2 (Arg-Pro-
Pro-Gly-Phe-Ser-Pro-Phe, m/z 905), was monitored as a function of
time. Solid lines are fits to the experimental data.
shows any accumulation of an EP2 complex. The same observation
was made in experiments that used different reaction times,
different substrate concentrations, and samples that had a different
ratio of R- to δ′-chymotrypsin. The absence of an observable EP2
complex in this case is not due to a lack of enzyme activity; on
the contrary, it will be seen that the enzyme undergoes rapid
turnover under the conditions of Figure 4 (see below). It has
previously been established that in the case of peptide bond
hydrolysis by chymotrypsin, k3 is much larger than k2. In other
words, acylation of the enzyme is the rate-determining step in
Scheme 1 under these conditions.1-3,45,46 EP2 is being formed
slowly and hydrolyzed quickly, and therefore, it does not become
significantly populated at any point in the reaction. A pre-steady-
state analysis, based on the concepts used above, is not possible
under these conditions. Instead, it will be demonstrated how the
ESI-MS-coupled capillary mixing setup can be applied to study
the reaction kinetics under steady-state conditions.
The formation of P2 was monitored at different bradykinin
concentrations. Typical intensity-time profiles are depicted in
Figure 5, together with the corresponding linear fits. As predicted
by eq 6, the reaction rate increases with increasing substrate
concentration. An unexpected feature of Figure 5 is the observa-
tion of a concomitant increase of the initial signal intensities I0.
This effect is caused by the presence of a small amount of P2 as
an impurity in the commercially supplied bradykinin substrate. A
plot of I0 for different bradykinin concentrations is depicted in
Figure 6A. This figure shows a linear increase of I0 up to substrate
concentrations of ∼2 mM. Surprisingly, this is followed by a range
where I0 decreases with increasing bradykinin concentration. This
observation is attributed to a suppression of P2 ions, caused by
the very high concentration of bradykinin in the solution. Effects
of this kind are a well-known occurrence in ESI-MS.47,48
Fig u re 6 . Data obtained for the hydrolysis of bradykinin by
chymotrypsin, determined from kinetic profiles similar to those
depicted in Figure 5. (A) Dependence of the initial product intensity,
I0, on the bradykinin concentration. Open symbols for concentrations
of 3, 4, and 5 mM correspond to values corrected for signal
suppression effects. These corrected data points are based on a linear
extrapolation of the I0 values measured at bradykinin concentrations
of up to 2 mM (solid line). (B) Michealis-Menten plot of the reaction
rate vs substrate concentration. Closed symbols represent measured
values. Open symbols correspond to reaction rates adjusted for signal
suppression; they were obtained by multiplication of the measured
values with correction factors obtained from panel A. Slopes of the
measured ESI-MS intensity profiles (in units of counts/s) were
converted to reaction rates (in units of M s-1) by using a conversion
factor of 84 counts/s µM-1. This factor was determined in separate
experiments, using standardized solutions of purified P2 (ꢀ258 ) 390
M-1 cm-1). The solid line in (B) is a fit to the corrected data set based
on eq 6.
The dependence of the reaction rate on the bradykinin
concentration was determined from the measured ESI-MS kinetic
profiles, resulting in the data depicted in Figure 6B. The measured
rates increase up to a substrate concentration of 2 mM, followed
by a decrease. This decrease is ascribed to the same signal
suppression effect discussed for I0. Using the P2 impurity in the
bradykinin solution as an internal calibrant, the measured reaction
rates were corrected for this effect, employing the procedure
(45) Brandt, K. G.; Himoe, A.; Hess, G. P. J. Biol. Chem. 1 9 6 7 , 242, 3973-
3982.
(46) Himoe, A.; Brandt, K. G.; Hess, G. P. J. Biol. Chem. 1 9 6 7 , 242, 3963-
3972.
(47) Pan, P.; McLuckey, S. A. Anal. Chem. 2 0 0 3 , 75, 1491-1499.
(48) Pan, P.; McLuckey, S. A. Anal. Chem. 2 0 0 3 , 75, 5468-5474.
2542 Analytical Chemistry, Vol. 76, No. 9, May 1, 2004

outlined in the caption of Figure 6. Thus, a Michaelis-Menten
plot was produced (Figure 6B), from which the steady-state
parameters KM ) 0.51 ( 0.08 mM and kcat ) 43 ( 2 s-1 were
determined, resulting in a specificity constant of kcat/ KM ) 8.4 ×
activity of coexisting enzyme species is reminiscent of recently
developed approaches for monitoring the thermodynamic stabili-
ties of unpurified proteins in mixtures by hydrogen/ deuterium
exchange methods and MS.50 In both cases, advantage is taken
of the inherent selectivity of MS-based techniques.
104 s-1 M-1
.
Given the fact that bradykinin is a nonchromophoric substrate,
it is not surprising that there seems to be a lack of literature data
for direct comparison with the steady-state kinetics reported here.
DelMar et al.49 compiled parameters for a number of chro-
mophoric oligopeptide substrate analogues of chymotrypsin. Many
of these compounds show KM values in the range around 0.5 mM,
which is consistent with our results. The kcat values of those
substrate analogues show a large spread, from 0.01 s-1 up to more
Both “spectral” and “kinetic” MS data are required for the
characterization of enzymatic reaction mechanisms. Measure-
ments in spectral mode allow an analysis of the components of
the reaction mixture, in particular the identification of mechanisti-
cally important intermediates. Data recorded in kinetic mode
provide the rate constant(s) of the reaction. Previous MS-based
techniques were capable of providing data in either kinetic20,21 or
spectral mode,12,23,27 but not both. In principle, it is possible to
obtain intensity-time profiles for the determination of rate
constants by “piecing together” spectral mode data.24 However,
that approach is very tedious, and the resulting kinetic traces
consist of only relatively few data points. It is an inherent
advantage of the ESI-MS-coupled capillary mixing setup used here
that it is capable of providing mass spectra recorded at selected
reaction times, as well as detailed intensity-time profiles with an
extremely high density of data points.
For this study, a key modification was introduced to our
previously described instrumental setup.29 By using a second
mixing step at the outlet of the reaction capillary it is possible to
add a makeup solvent to improve the signal intensity and stability
in ESI-MS. In the present case, meaningful kinetic experiments
without this modification would not have been possible. The
addition of acid in the current experiments leads to quenching of
the reaction, ∼30 ms before ionization occurs. Unfortunately, the
use of these non-native solution conditions at the ESI source likely
precludes the observation of noncovalently bound intermediates,
such as the ES complex in Scheme 1. One limitation of the current
on-line ESI-MS method is its incompatibility with high concentra-
tions of salts, buffers, and other solvent additives that have
detrimental effects on the ionization process.51 We are confident
that it will be possible to overcome this problem in future studies,
by including a rapid on-line desalting device between the outlet
of the mixing capillary and the inlet of the ESI source. It is hoped
that modifications of this kind will further broaden the applicability
of this method to study the mechanisms of various (bio)chemical
processes.
than 100 s-1 and specificity constants between 10 and 107 s-1 M-1
.
The corresponding results obtained in the current study for a
“natural” chymotrypsin substrate, therefore, are located in the
midrange of the parameters determined for those chromophoric
compounds.
CONCLUSIONS
This work employs a newly developed rapid mixing apparatus
for time-resolved studies by ESI-MS. This methodology opens a
wide area for kinetic studies on enzyme reactions, without
requiring chromophoric or radioactively labeled substrates. It is
anticipated that future uses of this approach will be particularly
powerful for pre-steady-state kinetic measurements, because these
experiments provide direct information on the reaction mecha-
nisms of enzyme-catalyzed reactions. In cases where pre-steady-
state measurements are not feasible, as for bradykinin, ESI-MS-
based studies can still provide valuable kinetic information based
on steady-state measurements.
Signal suppression artifacts represent a potential problem in
assays that monitor the release of a reaction product at different
substrate concentrations. The present work illustrates a simple
method to correct for this effect. In the case of bradykinin, it was
fortuitous that the substrate contained a trace amount of reaction
product that could be used as an internal calibrant. In other cases,
it may be necessary to add such a calibrant to the reaction mixture.
It is noted that potential suppression effects are of no concern for
the pre-steady-state experiments on p-NPA, because the measured
kobs values do not depend on absolute signal intensities.
An interesting feature of the ESI-MS-based method used in
this study is the potential to measure the kinetics for individual
components of an enzyme mixture. This was demonstrated in the
p-NPA experiments, where the accumulation of two different
reaction intermediates, corresponding to the R and δ′ forms of
chymotrypsin, could be monitored. The capability to probe the
ACKNOWLEDGMENT
We thank Paula Pittock from Dr. Gilles Lajoie’s laboratory for
help with the chromatographic purification of the bradykinin
hydrolysis product P2. Dr. Jagan M. Payidiparty was involved in
preliminary studies on p-NPA hydrolysis. This work was financially
supported by the Natural Sciences and Engineering Research
Council of Canada (NSERC), the Canada Foundation for Innova-
tion (CFI), and the Provincial Government of Ontario.
(49) DelMar, E. G.; Largman, C.; Brodrick, J. W.; Geokas, M. C. Anal. Biochem.
1 9 7 9 , 99, 316-320.
(50) Ghaemmaghami, S.; Fitzgerald, M. C.; Oas, T. G. Proc. Natl. Acad. Sci. U.S.A.
2 0 0 0 , 97, 8296-8301.
(51) Xu, N.; Lin, Y.; Hofstadler, S. A.; Matson, D.; Call, C. J.; Smith, R. D. Anal.
Chem. 1 9 9 8 , 70, 3553-3556.
(52) Cunniff, J. B.; Vouros, P. J. Am. Soc. Mass Spectrom. 1 9 9 5 , 6, 437-447.
(53) Collings, B. A.; Douglas, D. J. J. Am. Chem. Soc. 1 9 9 6 , 118, 4488-4489.
Received for review December 23, 2003. Accepted
February 26, 2004.
AC0355348
Analytical Chemistry, Vol. 76, No. 9, May 1, 2004 2543