Isothermal titration calorimetric study of RNase-A kinetics (cCMP → 3′-CMP) involving end-product inhibition

Article abstract of DOI:10.1023/B:PHAM.0000041460.78128.0f

Purpose. Isothermal titration calorimetry (ITC) and progress curve analysis was used to measure the enzyme kinetic parameters (K _M and k _cat) of the hydrolysis of cCMP by RNase-A, a reaction that includes end-product competitive inhibition by 3'-CMP. Methods. The heat generated from injection of 9-15 μl cCMP (20 mM) into bovine pancreatic RNase-A (600 nM) in 50 mM Na⁺ acetate buffer (pH 5.5; 37°C) was monitored for 1500-2000 s. Thermal power (dQ/dt), equal to (1) /ΔH _app × d(cCMP)/dt was recorded every 1 s. The end-product inhibition constant (K _p) and enthalpy of the inhibitor binding interaction was obtained from the saturation data of 60 sequential injections of 3′-CMP (1.2 mM) into 0.05 mM RNase-A. The data of the plot of -d[cCMP]/dt against [cCMP] were fitted to kinetic equations incorporating K _p to yield K _M and k _cat. Results. ΔH _app for each run was obtained by integration of the progress curve. The plot of -d[cCMP]/dt against [cCMP] yielded the kinetic parameters K _M = 105.3 μM, 121.6 μM, and 131.3 μM; k _cat = 1.63 s^-1, 1.56 s^-1, and 1.71 s^-1. The end-product bound with 1:1 stoichiometry and K _p = 53.2 μM. Conclusions. The combination of progress curve analysis and ITC allowed rapid and facile measurement of the kinetic parameters for catalytic conversion of cCMP to 3′-CMP by RNase-A, a reaction complicated by end-product inhibition.

Full text of DOI:10.1023/B:PHAM.0000041460.78128.0f

Pharmaceutical Research, Vol. 21, No. 9, September 2004 (© 2004)
Research Paper
“transphosphorylation” reaction from the 5Ј carbon to the 2Ј
carbon of the next nucleotide in the RNA chain. Specific
amino acid residues of the RNase form the catalytic reaction
domain, the details of which have been determined by several
strategies, including chemical modification and site-directed
mutagenesis studies (e.g., 12–15). In the second step, the sub-
ject of the present report, the product of the first step (2Ј,3Ј-
cyclic phosphodiester) is hydrolyzed to a 3Ј nucleotide (3Ј-
CMP) (16,17) (Fig. 1). Both of these reactions are catalyzed
by RNase. A complication in obtaining kinetic parameters for
this reaction arises because 3Ј-CMP is an inhibitor of
RNase-A and, thus, results in end-product inhibition of the
overall reaction.
Isothermal Titration Calorimetric
Study of RNase-A Kinetics (cCMP →
3؅-CMP) Involving
End-Product Inhibition
Shawn D. Spencer1 and Robert B. Raffa1,2,3
Received November 20, 2003; accepted May 17, 2004
Purpose. Isothermal titration calorimetry (ITC) and progress curve
analysis was used to measure the enzyme kinetic parameters (KM and
kcat) of the hydrolysis of cCMP by RNase-A, a reaction that includes
end-product competitive inhibition by 3Ј-CMP.
Methods. The heat generated from injection of 9–15 ␮l cCMP (20
mM) into bovine pancreatic RNase-A (600 nM) in 50 mM Na+ ac-
etate buffer (pH 5.5; 37°C) was monitored for 1500–2000 s. Thermal
power (dQ/dt), equal to (1) /⌬Happ × d(cCMP)/dt was recorded every
1 s. The end-product inhibition constant (Kp) and enthalpy of the
inhibitor binding interaction was obtained from the saturation data of
60 sequential injections of 3Ј-CMP (1.2 mM) into 0.05 mM RNase-A.
The data of the plot of –d[cCMP]/dt against [cCMP] were fitted to
Isothermal titration (micro)calorimetry (ITC) is a highly
accurate and automated technique that directly measures the
apparent enthalpy change (⌬Happ) associated with reactions
in solution and it is used to obtain thermodynamic parameters
for ligand-macromolecule interations (18). In the standard
ITC apparatus, the reaction proceeds in a sample cell of rela-
tively small volume (usually 1–2 ml). One component of the
reaction is already in the reaction cell (e.g., enzyme), the
other component (e.g., substrate or inhibitor) is added by an
automated injection system in measured amounts for mea-
sured times. A stirrer ensures that the reaction is continuously
and well mixed. The reaction cell is composed of material of
good thermal conductivity such that energy changes occurring
within the reaction cell are transmitted as temperature
changes. In modern ITC equipment the change is measured
as the differential current (power) that is required to maintain
the reaction cell at the same pre-set temperature of a refer-
ence cell. Thus, the measurements are highly precise and re-
producible. Following each injection, the power is recorded as
a function of time. If the reaction is exothermic, less thermal
power (J s–1) is required relative to the reference cell; if the
reaction is endothermic, more power is required (the total
system is closely maintained at a preset temperature). Details
about ITC are available in several excellent reviews (19–21).
Todd and Gomez (22) have described techniques for de-
termining enzyme kinetic constants using ITC based on the
proportionality between the rate of a reaction and the ther-
mal power generated, but did not examine RNase. Cai et al.
(23) used ITC to examine RNase-A, but used the initial rate
approximation. Koerber and Frank (24) examined hydrolysis
of 3Ј-CMP by RNase-A using a new method based on nu-
merical differentiation of the complete reaction (progress)
curve in which data are fitted to the rate equation itself (25)—
which they showed had advantages over initial velocity or
integrated Michaelis-Menten equation methods—but mea-
sured enzyme activity spectrophotometrically. The present
study is the first, to our knowledge, to combine the advan-
tages of ITC and differential analysis of the progress curve to
the enzymatic conversion of cCMP to 3Ј-CMP by RNase-A,
which involves end-product inhibition by 3Ј-CMP.
kinetic equations incorporating Kp to yield KM and kcat
.
Results. ⌬Happ for each run was obtained by integration of the prog-
ress curve. The plot of –d[cCMP]/dt against [cCMP] yielded the ki-
netic parameters KM ס 105.3 ␮M, 121.6 ␮M, and 131.3 ␮M; kcat
ס
1.63 s−1, 1.56 s−1, and 1.71 s−1. The end-product bound with 1:1 stoi-
chiometry and Kp ס 53.2 ␮M.
Conclusions. The combination of progress curve analysis and ITC
allowed rapid and facile measurement of the kinetic parameters for
catalytic conversion of cCMP to 3Ј-CMP by RNase-A, a reaction
complicated by end-product inhibition.
KEY WORDS: calorimetry; end-product inhibition; enzyme kinet-
ics; RNase.
INTRODUCTION
RNases (ribonucleases) constitute a family of cellular
exo- and endonuclease [EC 3.1.27.5] enzymes that are present
in vertebrates, bacteria (1,2,3), mold (4), and plants (5,6).
They perform important functions in the processing of RNA,
which has been estimated to comprise about 20% of a cell’s
dry mass (1). However, RNases can be overtly toxic (7) or can
exacerbate pathologic conditions. For example: angiogenin,
an RNase found in plasma, induces angioneogenesis in tu-
mors (8); RNase-2 (RNase U) is neurotoxic and may produce
some of the symptoms associated with overproduction of eo-
sinophils (9); and bovine seminal RNase is immunosuppres-
sive, embryotoxic, and aspermatogenic (10). Hence, either
activators or inhibitors of RNases could have therapeutic po-
tential as novel pharmaceutical agents.
RNases display a preference for pyrimidine bases (cyto-
sine and uracil) of RNA. The catalytic mechanism of RNA
cleavage by RNases is thought to occur in two steps (11). In
the first step, a 2Ј,3Ј-cyclic phosphodiester is formed by a
MATERIALS AND METHODS
Materials
1 Department of Pharmaceutical Sciences, School of Pharmacy,
Temple University, Philadelphia, Pennsylvania 19140, USA.
2 Department of Pharmacology, School of Medicine, Temple Univer-
sity, Philadelphia, Pennsylvania 19140, USA.
Bovine pancreatic RNase (RNase-A, type XII-A, EC
3.1.27.5), cCMP (2Ј,3Ј- CMP; 2Ј,3Ј-cyclic cytidine monophos-
phate), and 3Ј-CMP were purchased from Sigma-Aldrich (St.
Louis, MO). Buffer chemicals, analytical grade, were pur-
3 To whom correspondence should be addressed. (e-mail: robert.
raffa@temple.edu)
0724-8741/04/0900-1642/0 © 2004 Springer Science+Business Media, Inc.
1642

Enzyme Kinetics of Hydrolysis by RNase-A Using ITC
1643
d S d P
͓ ͔
͓ ͔
−
=
,
(2)
dt
dt
and the amount of heat associated with converting n moles of
substrate into product:
Q = n и ⌬Happ = P total и V и ⌬Happ
͓ ͔
.
(3)
The magnitude of the experimentally-measured apparent
molar enthalpy change (⌬Happ) approaches the magnitude of
the intrinsic enthalpy change (⌬Hint) when the reaction is
conducted in buffers that have a small heat of ionization
(⌬Hion) such as acetate (26), since ⌬Happ ס ⌬Hint + ␩⌬Hion
where ␩ is the number or protons released from the buffer. A
plot of –d[S]/dt against [S] (i.e., –d[cCMP]/dt against [cCMP])
yields the kinetic parameters from the steady-state rate equa-
tion for the general case of noncooperative, single-substrate,
single-product enzymatic catalysis (24):
VM S
͓ ͔
KM
ͩ
ͪ
1 −
Kp
d P
͓ ͔
ր
p
=
,
(4)
dt
S
+ K
͔
͓
0
+ S
͓ ͔
K
− 1
ͩ
ͪ
M
KP
ր
where the end-product affinity (Kp) can either be estimated
as a fitting parameter or, as done in the present experiments,
can be obtained from a separate determination of the binding
of end-product (3Ј-CMP) with enzyme (RNase-A).
Fig. 1. Reaction scheme for the two-step catalytic action of RNase-A.
RNA is the substrate for the first transphosphorylation reaction, and
2Ј,3Ј-cyclic CMP (cCMP) is the product which, in turn, is the substrate
for the second hydrolysis reaction that produced 3Ј-CMP.
Procedure
The ITC instrumentation (VP-ITC Microcal, LLC,
Northampton, MA) has been described previously (27). In
short, the data collection involves monitoring the electrical
power supplied to the compensation heater attached to the
reaction cell that is required to maintain isothermal condi-
tions. Thus, the measured experimental quantity is the energy
(heat) flow from the sample cell, which is directly propor-
tional to the heat of the reaction. Following an injection of
reactant into the reaction cell, the power supplied (or con-
served) to compensate for the reaction heat is evident as a
departure from baseline, attainment of a maximum excursion,
and return to baseline once catalysis is complete. The trap-
ezoidal rule yields the area under the curve between two data
points that were collected every second, and integration
yields the total AUC, which is Q for the reaction. The ratio
AUC/n, where n is the number of moles of substrate injected,
chased from VWR Scientific (Bridgewater, NJ). The micro-
calorimeter (model VP-ITC) was purchased from MicroCal
LLC (Northampton, MA).
Kinetic Parameters
The reaction mechanism for the interaction between
RNase-A (E) and cCMP (S) to form 3Ј-CMP (P) is repre-
sented by:
k
1
k
cat
E + S ⇔ ES → E + P,
k
−1
yields ⌬Happ
.
where kcat is the catalytic rate (substrate turnover rate) con-
stant and (k−1 + kcat)/k1 is the Michaelis-Menten constant
(KM). In a calorimeter, the rate of a reaction is related to the
measured heat by the following equation (22):
RNase-A and cCMP solutions were degassed under
vacuum for 10 min. A 250 ␮L syringe was filled with cCMP
(20 mM) dissolved in 50 mM Na+-acetate buffer (pH 5.5). The
1.4 ml reaction vessel (maintained at 37°) was filled with
RNase-A (600 nM, determined spectrophotometrically at 280
nm using ␧ ס 9800 cm−1 m−1) in 50 mM Na+-acetate buffer
(pH 5.5) and stirred at 270 rpm. The result of single injections
d P
͓ ͔
d S
͓ ͔
1
dQ
Rate =
= −
=
и
,
(1)
dt
dt
V и Happ dt
where [S] and [P] are the molar concentrations of the sub- of 9–15 ␮l was monitored for 1500–2000 s (to allow time for
strate (cCMP) and product (3Ј-CMP), respectively, V is the substrate depletion) and the thermal power (dQ/dt) in watts
volume of the reaction cell, ⌬Happ is the (apparent) molar was recorded every 1 s. The data were fit to kinetic equations
enthalpy for the reaction, and dQ/dt is the thermal power, using WINNONLIN (Scientific Consulting, Inc., Mountain
which is the rate of change of heat (Q) with respect to time (t). View, CA). The affinity constant of the 3Ј-CMP end-product
Equation 1 is obtained from the rate equality:
(Kp) was obtained using 60 injections of 3Ј-CMP (1.2 mM)

1644
Spencer and Raffa
into 0.05 mM RNase-A using methods described for the bind-
ing of 2Ј-CMP in prior publications (28,29).
RESULTS
End-Product Binding
The selected conditions produced heats for the interac-
tion 3Ј-CMP + RNase-A ⇔ 3Ј-CMP–RNase-A that yielded
consistent isotherms such as that shown in Fig. 2 (top). A
stable baseline was maintained over the course of the 60 in-
jections of 3Ј-CMP (1.2 mM) into RNase-A (0.05 mM). Each
downward deflection in the figure corresponds to heat gen-
eration of the exothermic reaction. The progressively smaller
heat output of each injection corresponds to saturation of
RNase-A binding sites by 3Ј-CMP. The heat of dilution was
determined from 10 additional injections into saturated en-
zyme (same result as into buffer), and was subtracted from
the reaction heat. The total time for each of the 3 replicate
runs was 6 h (60 injections × 360 s per interval). The maximal
output was generally 1.5–2 ␮cal s−1 (6–8 ␮J s−1). The data
were processed and deconvoluted using ORIGIN software (v
5.0, MicroCal Software, Northampton, MA). The transposed
data were normalized per mole of 3Ј-CMP injected by inte-
gration and were plotted as the integrated heats (Fig. 2, bot-
tom). A curve was obtained by computer-generated fit to a
Fig. 3. Representative isotherm monitored for 1500 s following a
single injection of cCMP (20 mM) into 600 nM of RNase-A at 37°C
(pH 5.5).
single-site binding model using nonlinear regression. From
the fitting parameters a stoichiometry of 1:1 was obtained
with Kp ס 53.2 1.5 ␮M and ⌬Happ ס −17.7 1.1 kcal mol−1
(–74.0 4.6 kJ mol−1).
Kinetics
Single injection of cCMP (20 mM) into RNase-A (600
nM) produced heat from the catalytic conversion cCMP →
3Ј-CMP that was detected as a downward deflection in the
isotherm, indicative of an exothermic interaction (Fig. 3).
Maximum heat output was obtained between 60 and 80 s after
injection. The system returned to baseline, indicative of com-
plete conversion of substrate (cCMP) to product (3Ј-CMP),
between 1500 and 2000 s. Conversion followed apparent first-
order reaction kinetics, as shown by a plot of the decline of
cCMP and increase of 3Ј-CMP with time (Fig. 4).
A series of 35 injections each yielded ⌬Happ according to
the integrated form of Eq. 1,
1
t
⌬Happ
=
и
dQ,
(5)
͐
t=0
cCMP 0 и V
͓
͔
which gave substrate concentration as a function of time ac-
cording to:
t
Q
͐
t=0
Fig. 2. Representative isotherm for the binding of 3Ј-CMP to RNase-
A. Top: raw data output of power (heat released) for each of 60
consecutive injections of 3Ј-CMP (1.2 mM) into RNase-A (0.05 mM)
at 37°C (pH 5.5). Bottom: heat exchange at each injection obtained
by integration of each injection “spike” in the top panel, normalized
to kcal mol−1 of 3Ј-CMP. The line is the computer-generated best-fit
to a single-site binding model.
[cCMP] = [cCMP]0 − [3Ј − CMP = [cCMP]0 −
.
(6)
V и ⌬Happ
The graph of d[3Ј-CMP]/dt is shown in Fig. 5. The data
were fitted to Eq. 4 using WINNONLIN (Scientific Consult-
ing, Inc., Mountain View, CA) software, yielding KM and kcat
as fitted parameters (Table I).

Enzyme Kinetics of Hydrolysis by RNase-A Using ITC
1645
Table I. Kinetic Constants (KM and kcat) for Enzymatic Conversion
of cCMP to 3Ј-CMP by RNase A Determined Using ITC and Prog-
ress Curve Analysis
140 ␮M cCMP
185 ␮M cCMP
230 ␮M cCMP
KM
kcat
105.3 12.1
1.63 0.25
121.6 14.5
1.56 0.02
131.3 9.3
1.71 0.07
Values are the means
SD of three independent measurements
(37°C, pH 5.5).
are important enzymes to study—and potential modulators
are being designed (35). RNase-A is the prototypic and most
commonly studied representative of the RNase family.
Enzyme kinetics equations are usually formulated in
terms of the dependence of reaction rate upon the concen-
tration of substrate, whereas most studies measure the
amount of substrate depletion or product generation. The
usual way this is resolved is to analytically differentiate the
data at zero-time and measure the initial rate of the reaction.
Because instantaneous velocity is generally not possible to
measure, it is necessary to define conditions where the prog-
ress curve is approximately straight for a measurable period
of time. Since the reaction rate is maximum at the initiation of
the reaction, the initial reaction rate is usually chosen. At less
than about 1–5% reaction progress, and under appropriate
experimental conditions, the initial-rate approximations yield
adequate measurements for enzymatic biochemical reactions
(36). However, for more complicated reactions, such as those
that generate products that significantly inhibit enzyme activ-
ity (end-product inhibition), the practical requirements and
interpretation become more restrictive. It is sometimes pref-
erable that instead of initial rate analysis, the reaction “prog-
ress curve” can be differentiated or integrated and then fitted
to the appropriate, usually Michaelis-Menten, model equa-
tion. In the integral method, continuous data (i.e., [P] vs.
time) are fitted to an integrated rate equation; in the differ-
ential method, the experimental data, that is,, rate versus [S]
(“progress curve”) are fitted to a rate equation without trans-
formation. Both continuous methods offer the advantage of
utilizing the greater amount of the kinetic information con-
tained in the entire progress curve, although such techniques
can be computationally difficult. However, numerical differ-
ential methods for enzyme kinetic analysis, made practical
with the introduction of computer-assisted data acquisition of
large data-point collection, now allow accurate determination
even for reactions that exhibit strong product inhibition (24).
With the advent of modern power-compensated microcalo-
rimeters with fast response-time, equipment is now able to
take advantage of direct (not calculated) differential analysis.
This circumvents the use of the integrated Michaelis-Menten
equation and allows the advantages of full progress curve
analysis. ITC already is used successfully to measure the ther-
modynamic parameters of biochemical reactions (e.g., see re-
views 19, 20, 21), with application to drug-receptor interac-
tions (18,37). The goal of the present study was to extend our
ongoing study of RNase-A with ITC (28,29) to measure the
kinetic constants for the RNase-catalyzed conversion of
cCMP to 3Ј-CMP, a reaction with strong end-product inhibi-
tion.
Fig. 4. Same data as in Fig. 3 plotted as the time-dependent depletion
of substrate (cCMP) and generation of product (3Ј-CMP).
DISCUSSION
Members of the RNase superfamily have both intracel-
lular (‘non-secretory’) and extracellular (“secretory”) (30,31)
roles that contribute to their beneficial and desirable func-
tions. However, under certain circumstances, RNases can be
cytotoxic. An example is stimulation of blood vessel prolif-
eration (angioneogenesis) in tumors by the blood-borne
RNase angiogenin. Another is RNase-2, which is implicated
in conditions where eosinophils appear in excess numbers,
such as asthma and other inflammatory disorders in which
tissue damage occurs as part of an allergic response (e.g.,
32–34). For either its beneficial or deleterious roles, RNases
Fig. 5. Cumulative rate data for the catalytic hydrolysis of cCMP to
3Ј-CMP by RNase-A. The line is the best-fit computer-generated
nonlinear regression to Eq. 4.
The kinetic equations incorporating end-product inhibi-
tion include a term for the ⌬Happ of the binding of end-

1646
Spencer and Raffa
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RNase-A (28,29). The advantage of ITC is the ability to easily
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and the affinity constant Kp was 53.2 ␮M. The use of acetate
buffer enhances the accuracy of ⌬Happ determinations, be-
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fixed parameters into the kinetic equations allowed the de-
termination of the kinetic parameters KM and kcat from the
isotherms generated by injection of 3Ј-CMP into the reaction
cell containing RNase-A. In our experiment, the Michaelis
constant KM ס 105.3 ␮M, 121.6␮M, and 131.3␮M and the kcat
(turnover rate) ס 1.63 s−1, 1.56 s−1, and 1.71 s−1, depending
on the initial substrate concentration. As expected, the turn-
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centration. These values are in close agreement with the only
comparative literature values (KP ס 72 ␮M, kcat ס 2.2 s−1
,
and KM ס 820 ␮M) (39). The differences are probably at-
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product inhibition. The procedure used differential analysis
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parameter estimation and uses all of the information in a
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