COMM-4039; No.

of Pages 6
ARTICLE IN PRESS
c o m p u t e r m e t h o d s a n d p r o g r a m s i n b i o m e d i c i n e x x x ( 2 0 1 5 ) xxx–xxx

journal homepage: www.intl.elsevierhealth.com/journals/cmpb

Enzyme inhibition studies by integrated

Michaelis–Menten equation considering
simultaneous presence of two inhibitors when one
of them is a reaction product

Rui M.F. Bezerra ∗ , Paula A. Pinto, Irene Fraga, Albino A. Dias

Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas, CITAB, Universidade de Trás-os-Montes e
Alto Douro, UTAD, 5000-801 Vila Real, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: To determine initial velocities of enzyme catalyzed reactions without theoretical errors
Received 6 July 2015 it is necessary to consider the use of the integrated Michaelis–Menten equation. When
Received in revised form the reaction product is an inhibitor, this approach is particularly important. Neverthe-
2 December 2015 less, kinetic studies usually involved the evaluation of other inhibitors beyond the reaction
Accepted 8 December 2015 product. The occurrence of these situations emphasizes the importance of extending the
integrated Michaelis–Menten equation, assuming the simultaneous presence of more than
Keywords: one inhibitor because reaction product is always present. This methodology is illustrated
Integrated Michaelis–Menten with the reaction catalyzed by alkaline phosphatase inhibited by phosphate (reaction prod-
equations uct, inhibitor 1) and urea (inhibitor 2). The approach is explained in a step by step manner
Linear inhibition using an Excel spreadsheet (available as a template in Appendix). Curve fitting by nonlinear
General mechanism kinetics regression was performed with the Solver add-in (Microsoft Office Excel). Discrimination
Diagnosis of enzyme inhibition of the kinetic models was carried out based on Akaike information criterion. This work
Two inhibitors presents a methodology that can be used to develop an automated process, to discriminate
in real time the inhibition type and kinetic constants as data (product vs. time) are achieved
by the spectrophotometer.
© 2016 Elsevier Ireland Ltd. All rights reserved.

be integrated to give a description of the reaction progress.

1. Introduction
The usual determination of initial velocities by drawing a
tangent line to the reaction curve is theoretically incorrect
since the substrate decrease and potential inhibitory reac-
tion products accumulate. As previously explained there is
an incompatibility between the kinetic data and the under-
lying model [1]. To avoid this incompatibility the model must
Another possibility is to differentiate the data (determining
rates) nevertheless if an appropriated kinetic model for a given
enzyme reaction is not known, determination of initial veloc-
ities is not theoretically accurate. Thus the kinetic studies
must evolve by the use of the integrated Michaelis–Menten
equations even if only for determination of initial velocities
[2,3].

∗
Corresponding author. Tel.: +351 259350465.
E-mail address: bezerra@utad.pt (R.M.F. Bezerra).
http://dx.doi.org/10.1016/j.cmpb.2015.12.013
0169-2607/© 2016 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated Michaelis–Menten equation consid-
ering simultaneous presence of two inhibitors when one of them is a reaction product, Comput. Methods Programs Biomed. (2015),
http://dx.doi.org/10.1016/j.cmpb.2015.12.013
COMM-4039; No. of Pages 6
ARTICLE IN PRESS
2 c o m p u t e r m e t h o d s a n d p r o g r a m s i n b i o m e d i c i n e x x x ( 2 0 1 5 ) xxx–xxx

The integrated Michaelis–Menten equation in its classi- program included in the Excel software that is only necessary
cal implicit form [4,5] or in its explicit form [6] have other to be activated to use them [5]) and discrimination between
advantages: (i) the initial addition of a product inhibitor can different models was performed by Akaike information cri-
be eliminated and Ki evaluated [7–10] allowing for example to terion (AIC) as previously explained [5,18]. Kinetic constants
determine inhibition constants, of a particular reaction prod- were also estimated by initial velocities [19]. Both kinetic
uct isomer, more accurately since they are based on correct methodologies used the same experimental data points.
stereochemistry and complete purity [11]; (ii) the possibility to Kinetic parameter uncertainties were determined by an Excel
determine the inhibition constants without knowledge of true template [22] and also by the software SPSS (Statistical Package
substrate concentration [9,12,13]. This is of special mention in for the Social Sciences).
heterogeneous reactions where the substrate is insoluble.
One of the main caveats regarding the use of integrated
Michaelis–Menten equation to entire progress curve analy- 3. Theory
sis is the possibility of gradual enzyme activity loss under
the assay conditions. However, nothing prevents its use with Kinetic analysis with the integrated Michaelis–Menten equa-
much lower percent substrate depletion [2,5,10]. Thus, in this tions permits the determination of inhibition constants (Ki )
work integrated equations will be applied only to data points when more than one reaction product inhibits the enzyme
usually utilized to determine initial velocities (up to 10% sub- [9,14,16]. In this situation:
strate depletion).
The integrated Michaelis–Menten equations have not been 
n
1 1
=
usually applied in the presence of two inhibitors in the reac- Kij Ki
j=1
tion medium. Nevertheless, integrated equations find special
interest in studies with two different inhibitors since prod- where Kij is the inhibition constant for one single product and
uct inhibition is often found as a common regulating enzyme Ki is the global inhibition constant taking into account all prod-
mechanism [4,9,14–17]. Thus, to study the inhibition kinetics ucts. In enzyme reactions characterized by the presence of two
of this kind of enzymes it is necessary to consider the simul- different product inhibitors, Hsu and Tsao [20] showed that if
taneous presence of two inhibitors because one of them is a there is no addition of an initial inhibitor (at time zero) the
reaction product (it is always present). The main objective of two constants, Kij1 and Kij2 cannot be individually determined
this work is to develop and apply integrated equations to the because they are in a lumped form. Nevertheless in the present
kinetic analysis of reactions carried out in the presence of two study a different condition occurs since urea is not a reaction
inhibitors when one of them is a reaction product whether or product which means that inhibition constants for urea and
not mutually exclusive. phosphate are not lumped [17]. Furthermore, Orsi and Tipton
[4] advocated that when two inhibitors exhibit different inhi-
bition types, linearization of integrated equations (e.g. [14]) is
2. Material and methods
not appropriate because different kinetic effect of inhibitors is
indistinctive. The use of non-linear regression is a simple way
2.1. Enzyme reagents and reaction conditions
to overcome linearization weakness [9].
All common types of linear inhibitions are included in
Alkaline phosphatase (E.C. 3.1.3.1.) was obtained from BDH.
linear mixed inhibition model (MI) since some inhibition
All the other reagents used are readily available from
constants can tend to infinity, meaning that they are irrelevant
major suppliers such as Merck. Stock solutions of 4-
giving rise to other linear models (see Supplement 1) [5,10].
nitrophenylphosphate disodium salt and 4-nitrophenol were
Assuming the mixed linear inhibition (MI) the following
prepared at concentrations of 20 mM. Reactions were car-
Michaelis–Menten rate equation is obtained:
ried out at 37 ◦ C containing 9.5 ␮g enzyme in 2.75 ml reaction
volume and different amounts of 4-nitrophenylphosphate
Vmax [S0 ]
(between 18.4 and 1500.0 ␮М) in 0.1 M Tris/HCl buffer, pH 9.0. v=     (1)
[I] [I]
The amount of product (4-nitrophenol) formed at several time Km 1 + Kic + [S] 1 + Kiu
intervals (10–60 s) was determined by absorbance at 405 nm,
using a spectrophotometer (path length, 1 cm) in conditions where Km , Michaelis constant; Kic , inhibitor dissociation con-
that never surpass 10% of substrate depletion. Another set of stant of enzyme-inhibitor complex; Kiu , inhibitor dissociation
similar reaction experiments was also performed but in the constant of enzyme-substrate-inhibitor complex; Vmax , max-
presence of 1.25 M urea (inhibitor). imum velocity of reaction; v, initial velocity of reaction; [I]
concentration of inhibitor and [S0 ] initial concentration of sub-
2.2. Data processing and analysis strate.
Considering the presence of two different inhibitors I1 and
Data processing and analysis was carried out by integrated I2 as well as different inhibition constants Kic1 and Kic2 or Kiu1
Michaelis–Menten equations according to [5] but taking into and Kiu2 the rate equation becomes:
account the simultaneous presence of two inhibitors being
one of them a reaction product (Supplement 1 and 2). Diagno- Vmax [S0 ]
v=     (2)
sis of enzyme inhibition and kinetic constants were estimated [I1 ] [I2 ] [I1 ] [I2 ]
Km 1 + Kic1 + Kic2 + [S] 1 + Kiu1 + Kiu2
by non-linear regression with Solver (Solver is an add-in

Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated Michaelis–Menten equation consid-
ering simultaneous presence of two inhibitors when one of them is a reaction product, Comput. Methods Programs Biomed. (2015),
http://dx.doi.org/10.1016/j.cmpb.2015.12.013
COMM-4039; No. of Pages 6
ARTICLE IN PRESS
c o m p u t e r m e t h o d s a n d p r o g r a m s i n b i o m e d i c i n e x x x ( 2 0 1 5 ) xxx–xxx 3

Now assuming that inhibitor I1 is also formed during the

Table 1 – Sum of square errors (SSE) values for different
reaction ([I1 ] = [Pt ] + [I1,0 ] where [S0 ] − [St ] = [Pt ]) and [St ] is the models.
concentration of substrate at the time t and the subscript “0”
WI CI NCI UCI MI
as S0 , I1,0 , means initial concentration of the I1 at t = 0, the rate
equation obtained is: SSE 3976.3 1455.2 468.7 1019.0 385.1
p 2 4 4 4 6
d[S] n 112 112 112 112 112
v=−
dt
function) has the advantage to preserve statistical rigor since
Vmax [S0 ]
=     (3) substrate concentration is a function of time [6]. Due to fitting
[I1,0 ] [Pt ] [I2 ] [I1,0 ] [Pt ] [I2 ]
Km 1 + Kic1 + Kic1 + Kic2 + [S] 1 + Kiu1 + Kiu1 + Kiu2 difficulties of the intrinsic W-Lambert function a new method
(logistic enzyme kinetics) was developed [23]. Nevertheless
the implicit form continues to be used and the methodology
In Eq. (3), inhibition constants Kic1 and Kic2 or Kiu1 and Kiu2
presented in this paper can be adapted to accommodate an
are in a lumped form but after integration assuming that Pt
explicit form.
increases with time, the inhibition constants cease to be in a
lumped form [9,21].
Rearranging and integrating, gives: 4. Results and discussion
 
[I1,0 ] [S0 ]−[St ] [I2 ] The utilization of Microsoft Office Excel software and
Km 1 + Kic1 + Kic1 + Kic2
  the Solver add-in to determine kinetic parameters of the
 t  [St ] +[S0 ] 1 +
[I1,0 ]
+
[S0 ]−[St ]
+
[I2 ] Michaelis–Menten equation has been previously explained
Kiu1 Kiu1 Kiu2
− dt = d[St ] (4) [5,19]. The only difference is that the equations are more com-
0 [S0 ]
Vmax [S0 ]
plex and there are two different concentrations of inhibitors.
To facilitate the insertion of equations in the appropriate Excel
Or
cell, they were partitioned into ˛, ˇ and  components as
 t   [St ] explained in Supplement 1 and 2. Briefly, it is necessary to fill
[S0 ] [I1,0 ] [I2 ] 1
− dt Vmax = Km 1 + + + d[St ] a blank worksheet with data points similarly to Excel spread-
0
Kic1 Kic1 Kic2 [S0 ]
[St ]
sheet (Supplement 2) and to calculate the time necessary to
 Km [S0 ] [I1,0 ]
 [St ]
obtain the product P (calculated time).
[I2 ]
+ 1− + + + d[St ]
Kiu1 Kiu1 Kiu1 Kiu2 This kinetic investigation consisted of two parts: dis-
[S0 ]
 1
 [St ]
crimination between available models (Tables 1 and 2) and
+ − [S]d[St ] (5) parameter estimation (Table 3). According to Table 1, mod-
Kiu1 [S0 ] els CI, NCI and UCI have the same number of parameters
and so it is enough to compare the SSE value for discrimi-
Carrying out the integration (assuming Vmax = [E]kv where nation. The comparison reveals that NCI is better than any
[E] is the concentration of the enzyme and kv is a velocity other model since the SSE value is lower. However, the lowest
constant) the Eq. (6) is obtained. SSE value was obtained with the MI model, but the increase in
parameter numbers rise the incertitude about the best model.
 1
  [S0 ] [I1,0 ] [I2 ]
 [St ] Thus, statistical discrimination is necessary to compare NCI
t=− − Km 1 + + + ln
[E]kv Kic1 Kic1 Kic2 [S0 ] with MI models. There are several methods to compare and
 Km [S0 ] [I1,0 ] [I2 ]
 discriminate models such as extra sum-of-square F test, as
+ 1− + + + ([St ] − [S0 ]) Mannervik [24] or Akaike information criterion (AIC) [25]. The
Kiu1 Kiu1 Kiu1 Kiu2
1
 1
  AIC methodology (Table 2) was chosen because it has the
+ − ([St ]2 − [S0 ]2 ) (6) advantage that kinetic models can be nested or non-nested
2 Kiu1
and the results give a continuous scale for model plausibility
as previously discussed [5,18]. The results show that the bet-
The Eq. (6) can be simplified, giving rise to simplified nested ter model is the MI model with a probability near 100% and
models (Supplement 2) with fewer number of parameters such should be preferred. For the same reason it is also necessary
as the uncompetitive inhibition (UC), noncompetitive inhibi- to compare the WI with MI (Table 2) in order to rule out that
tion (NCI), competitive inhibition (CI) or without inhibition model WI is not appropriated. Experimental data points and
(WI) [5,10]. These models are simplifications of model MI, simulation lines using model MI with Solver optimized param-
assuming that different constants (Kiu1 , Kic1 , Kiu2 , Kic2 ) tend to eters (Table 3) are shown in Fig. 1A. To compare using initial
infinity. In Supplement 1 the equations are also rewritten in velocities methodology [19] the values are presented in Fig. 1B
order to be used in Excel software accommodating one or two and Table 3. Vmax (␮mol min−1 mg−1 ) can be obtained from kv
inhibitors or even no inhibitor. These equations have a wider (␮M s−1 (␮g/2.75 ml)−1 ) [5]. The residual plots (Fig. 2) obtained
range of applicability than those used before [5] and should be from data in Fig. 1 show the absence of systematic errors
preferred. once the residuals are scattered above and below of predicted
These equations are always equations for predicting the curve.
reaction time it takes to obtain a certain amount of prod- By the analysis of Table 3 it is evident than Kiu1 (uncom-
uct. The explicit form (expressed in terms of the W-Lambert petitive inhibition constant of phosphate) tend to infinity (is

Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated Michaelis–Menten equation consid-
ering simultaneous presence of two inhibitors when one of them is a reaction product, Comput. Methods Programs Biomed. (2015),
http://dx.doi.org/10.1016/j.cmpb.2015.12.013
COMM-4039; No. of Pages 6
ARTICLE IN PRESS
4 c o m p u t e r m e t h o d s a n d p r o g r a m s i n b i o m e d i c i n e x x x ( 2 0 1 5 ) xxx–xxx

Table 2 – Summary of model discrimination using Akaike information criterion methodology in the presence of
phosphate and urea.
Models A/B SSEA SSEB n PA +1 PB +1 AICA AICB AICcA AICcB  Probability B correct
WI/MI 3976.3 385.1 112 3 7 405.8 152.3 406.0 153.4 −252.6 1.0
NCI/MI 468.7 385.1 112 5 7 170.3 152.3 170.9 153.4 −17.5 0.99984

Table 3 – Summary of obtained constants with the best model (MI model).
Michaelis–Menten Km (␮M) Kic1 (␮M) Kiu1 (␮M) Kic2 (M) Kiu2 (M) Vmax (␮mol min−1 mg−1 )
Integrated equation 41.4 ± 1.2 7.0 ± 1.5 3.3 × 106 ± 0.0 2.4 ± 0.3 3.3 ± 0.3 3.0 ± 0.0
Initial velocities 58.3 ± 2.1 3.4 ± 0.2 3.4 ± 0.2 3.0 ± 0.0

A 6
A
60

50 3

40
Time (s)

Residuals
30 0
0 15 30 45 60
20

10 -3

0
0 2 4 6 8 10
-6 Time (s)
Phosphate (µM)

B 3
0.12
B
µmol min-1 mg-1

2 0.06
Residuals

1 0
0 0.5 1 1.5 2 2.5 3

0 -0.06
0 300 600 900 1200 1500
4-Nitrophenyl phosphate (µM)
-0.12 v (µmol min-1 mg-1)
Fig. 1 – (A) Experimental data points and simulation lines
using model MI (integrated Michaelis–Menten equation) Fig. 2 – (A) Residual plot from data of Fig. 1A. (B) Residual
with optimized constants. Squares and continuous line plot from data of Fig. 1B. The plot patterns emphasize the
were obtained without inhibitor urea and circles and dash absence of systematic errors.
line were obtained in the presence of 1.25 mM urea. (B) Data
points (initial velocities) and simulation lines using model
NCI with optimized constants (Table 3). Squares were
obtained without inhibitor urea and circles were obtained Table 4 – Sum of square errors (SSE) values for different
models assuming only the assays in the absence of urea.
in the presence of 1.25 mM urea.
WI CI NCI UCI MI
SSE 1327.5 83.1 120.2 114.8 83.1
too large compared to Kic1 ) and therefore phosphate (inhibitor p 2 3 3 3 4
1) is predominately a competitive inhibitor. Urea (inhibitor 2) n 62 62 62 62 62
presents similar values for Kiu2 and Kic2 , showing the predom-
inance of non-competitive inhibition. If both inhibitors are of urea has a predominantly noncompetitive inhibitor behavior
the same type, for example competitive, it will be expected (or eventually mixed).
that the best model is the competitive type. In this case the Supplement 3 contains a summary of hypothetical kinetic
discrimination of kinetic models was finished and the param- inhibition combinations obtained from possible results of the
eters can be obtained from the best model discriminated. The best model discriminated. In the presence of different types
results of Table 3 do not fit in this situation because phos- of inhibition (as in this study), it is necessary to confirm inhi-
phate has a predominantly competitive inhibitor behavior and bition patterns of phosphate and urea individually.

Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated Michaelis–Menten equation consid-
ering simultaneous presence of two inhibitors when one of them is a reaction product, Comput. Methods Programs Biomed. (2015),
http://dx.doi.org/10.1016/j.cmpb.2015.12.013
COMM-4039; No. of Pages 6
ARTICLE IN PRESS
c o m p u t e r m e t h o d s a n d p r o g r a m s i n b i o m e d i c i n e x x x ( 2 0 1 5 ) xxx–xxx 5

Table 5 – Summary of model discrimination using Akaike information criterion methodology assuming only the assays
in the absence of urea.
Models A/B SSEA SSEB n PA +1 PB +1 AICA AICB AICcA AICcB  Probability B correct
WI/CI 1327.5 83.1 62 3 4 196.0 26.1 1964.4 26.8 −169.6 1.00
CI/MI 83.1 83.1 62 4 5 26.1 28.1 26.8 29.2 2.4 0.23

product it is not possible to study the inhibition of urea with-

Table 6 – Modifications in the MI model to evaluate the
inhibition pattern of the second (urea) inhibitor (see out the presence of phosphate. Thus, it is necessary to copy
text). MI equation model (Supplement 1) to five spreadsheets and
to give e.g. the following names to the five spreadsheets
MI model Changes to make Number of
modified in the MI model parameters (WIm , CIm , NCIm , UCIm and MIm ; when “m” means modified).
remaining Modifications of MI model are necessary because phosphate
inhibition constants (Kic1 and Kiu1 ) are now fixed values and
WIm Removing [I2 ]/Kic2 2
(G10/$B$3) and [I2 ]/Kiu2 it is not possible to do that with the equations of Supple-
(G10/$B$6) ment 1. Thus Kic1 (cell B2) and Kiu1 (cell B5) will be fixed by
CIm Removing [I2 ]/Kiu2 3 inserting in each spreadsheet the values obtained in previ-
(G10/$B$6) ous study without the presence of urea (Kic1 = 10.7 ␮M and
NCIm Changing [I2 ]/Kiu2 3 Kiu1 = ∞ (e.g. 3.3 × 106 ␮M obtained in Table 3), i.e. converting
(G10/$B$6) to [I2 ]/Kic2
them in non-variable parameters). Additionally, other minor
(G10/$B$3)
changes in each of the five spreadsheets should be performed
UCIm Removing [I2 ]/Kic2 3
(G10/$B$3) as explained in Table 6. Before carried out nonlinear regres-
MI Do not remove anything 4 sion, do not forget to remove in the option “by change the
cell” (Solver dialog box) the cells $B$2 (Kic1 ) and $B$5 (Kiu1 ).
Table 7 – Sum of square errors (SSE) values for different The discrimination result in Tables 7 and 8 show that urea
models assuming the modifications of Table 6 (in the is a mixed inhibitor with a probability of 57%. The constants
presence of phosphate and urea). obtained were Kic2 = 2.6 M and Kiu2 = 3.3 M.
WIm CIm NCIm UCIm MIm Data points and model MI simulation with solver opti-
mized constants (Table 3, integrated equation) are presented
SSE 4258.3 1456.0 402.3 1096.9 392.6
p 2 3 3 3 4 in Fig. 1A. The constants obtained with the integrated
n 112 112 112 112 112 Michaelis–Menten equation and by traditional methods (ini-
tial velocities) are compared in Table 3. It should be pointed
In this case to say categorically that the phosphate is a out that determination of inhibition constants, Kic1 and Kiu1 ,
competitive inhibitor it is necessary to analyze the results by the Michaelis–Menten equation (initial velocities) requires
considering only assays with phosphate (initially present or the initial addition of phosphate in another set of assays
not) as explained previously [10]. In this study there is not without urea. As above explained, the study of urea inhi-
phosphate initially present but only generated as a reaction bition alone by initial velocities is not theoretically possible
product that increases with time. To do this is necessary to because phosphate is a reaction product. Several authors con-
analyze the experimental points obtained without the pres- cluded that urea is a noncompetitive inhibitor of alkaline
ence of urea (in Excel template are only considered the values phosphatase [26,27]. Nevertheless, in this work, the proba-
listed until the line 72) although used equations are the same. bility of alkaline phosphatase inhibition by urea to be of the
In this situation when nonlinear regression is performed, the noncompetitive type is not fully supported because slightly
quotients G10/$B$3 and G10/$B$6 have no influence on the different inhibition constants (Kic2 and Kiu2 ) were obtained
equation because cell G10 is always zero (cells $B$3 (Kic2 ) and in the MI model discriminated (Table 8) by Akaike informa-
$B$6 (Kiu2 ), do not change because they do not interfere in the tion criterion. This difference could be due to the fact that
equation). The results obtained in this situation are showed effect of phosphate inhibition was not considered in tra-
in Tables 4 and 5 and it is possible to conclude that CI is bet- ditional methods by initial velocities. Now it is possible to
ter than MI with a probability of 77%, and also better than WI state that phosphate is a competitive inhibitor and urea is
with a probability of 100%. The inhibition constant obtained a mixed inhibitor with the inhibition constants presented in
was Kic1 = 10.7 ␮M. Table 3.
In order to evaluate the inhibition pattern of urea, only It is important to point out that some alkaline phosphatase
a modified MI equation is used. As phosphate is a reaction isoenzymes present a reversible inhibition by urea (up to 2 M)

Table 8 – Summary of model discrimination using Akaike information criterion methodology assuming the
modifications of Table 6 (in the presence of phosphate and urea).
Models A/B SSEA SSEB n PA +1 PB +1 AICA AICB AICcA AICcB  Probability B correct
NCIm /MIm 402.3 392.6 112 4 5 150.5 148.4 151.6 151.1 −0.54 0.57

The subscript m in NCIm /MIm denotes that are modified MI equations as explained in text.

Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated Michaelis–Menten equation consid-
ering simultaneous presence of two inhibitors when one of them is a reaction product, Comput. Methods Programs Biomed. (2015),
http://dx.doi.org/10.1016/j.cmpb.2015.12.013
COMM-4039; No. of Pages 6
ARTICLE IN PRESS
6 c o m p u t e r m e t h o d s a n d p r o g r a m s i n b i o m e d i c i n e x x x ( 2 0 1 5 ) xxx–xxx
and others are more sensitive and present an irreversible inhi-
bition by denaturation [26,28], a phenomenon not observed
in this study. The use of the integrated Michaelis–Menten
equation to the entire progress curve is known for long time.
The option to use only initial data range was chosen in order
to theoretically eliminate possible interferences such as,
enzyme denaturation or change of initial conditions (pH,
temperature, etc.).
5. Conclusions
This work put in evidence that integrated Michaelis–Menten
equation is a powerful methodology for the analysis of enzyme
reactions even in the presence of two inhibitors being one
of them a reaction product. The methodology can be used
with only limited data points usually utilized to determine ini-
tial velocities. The possibility of utilizing this methodology to
determine kinetic constants in general kinetic studies and the
possibility of developing spectrophotometer software to indi-
cate “on line” and in real time the type of inhibition and kinetic
constants is important to point out.
Conflict of interest
None.
Acknowledgment
Authors are grateful for the financial support by CITAB through
project FCOMP-01-0124-FEDER-022692.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.cmpb.2015.12.013.
references
[1] R.G. Duggleby, Quantitative analysis of the time courses of
enzyme-catalyzed reactions, Methods 24 (2001) 168–174.
[2] A. Cornish-Bowden, The use of the direct linear plot for
determining initial velocities, Biochem. J. 149 (1975) 305–312.
[3] E.A. Boeker, Initial rates: a new plot, Biochem. J. 203 (1982)
(1982) 117–123.
[4] B.A. Orsi, K.F. Tipton, Kinetic analysis of progress curves,
Methods Enzymol. 63 (1979) 159–183.
[5] R.M.F. Bezerra, I. Fraga, A.A. Dias, Utilization of integrated
Michaelis–Menten equations for enzyme inhibition
diagnosis and determination of kinetic constants using
Solver supplement of Microsoft Office Excel, Comput.
Methods Programs Biomed. 109 (2013) 26–31.
[6] M. Golicnik, The integrated Michaelis–Menten rate equation:
deja vu or vu jade, J. Enzyme Inhib. Med. Chem. 28 (2013)
879–883.
[7] G.L. Atkins, I.A. Nimmo, The reliability of Michaelis
constants and maximum velocities estimated by using the
integrated Michaelis–Menten equation, Biochem. J. 135
(1973) 779–784.
Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated Michaelis–Menten equation consid-
ering simultaneous presence of two inhibitors when one of them is a reaction product, Comput. Methods Programs Biomed. (2015),
http://dx.doi.org/10.1016/j.cmpb.2015.12.013
[8] M. Markus, B. Hess, J.H. Ottaway, A. Cornish-Bowden, The
analysis of kinetic data in biochemistry. A critical evaluation
of methods, FEBS Lett. 63 (1976) 225–230.
[9] R.M.F. Bezerra, A.A. Dias, I. Fraga, A.N. Pereira, Simultaneous
ethanol and cellobiose inhibition of cellulose hydrolysis
studied with integrated equations assuming constant or
variable substrate concentration, Appl. Biochem. Biotechnol.
134 (2006) 27–38.
[10] R.M.F. Bezerra, A.A. Dias, Utilization of integrated
Michaelis–Menten equation to determine kinetic constants,
Biochem. Mol. Biol. Educ. 35 (2007) 145–150.
[11] R.G. Duggleby, J.F. Morrison, The analysis of progress curves
for enzyme-catalysed reactions by non-linear regression,
Biochim. Biophys. Acta 481 (1977) 279–312.
[12] H.N. Fernly, Statistical estimations in enzyme kinetics: the
integrated Michaelis–Menten, Eur. Biochem. J. 43 (1974)
377–378.
[13] S.L. Yun, C.H. Suelter, A simple method for calculating Km
and V from a single enzyme reaction progress curve,
Biochim. Biophys. Acta 480 (1977) 1–13.
[14] R.J. Foster, C. Niemann, The evaluation of the kinetic
constants of enzyme catalyzed reactions, J. Am. Chem. Soc.
77 (1955) 1886–1892.
[15] R.D. Philo, M.J. Selwyn, Use of progress curves to investigate
product inhibition in enzyme-catalysed reactions:
application to the soluble mitochondrial adenosine
triphosphatase, Biochem. J. 135 (1973) 525–530.
[16] R.M.F. Bezerra, A simple process to identify the type of
kinetics and estimate the kinetic parameters in the presence
of two different inhibitors, J. Med. Biochem. 3 (1999) 9–16.
[17] R.M.F. Bezerra, A.A. Dias, Enzymatic kinetic of cellulose
hydrolysis, inhibition by ethanol and cellobiose, Appl.
Biochem. Biotechnol. 126 (2005) 49–59.
[18] R.M.F. Bezerra, A.A. Dias, I. Fraga, A.N. Pereira, Cellulose
hydrolysis by cellobiohydrolase Cel7A shows mixed
hyperbolic product inhibition, Appl. Biochem. Biotechnol.
165 (2011) 178–189.
[19] A.A. Dias, P.A. Pinto, I. Fraga, R.M.F. Bezerra, Diagnosis of
enzyme inhibition using excel solver: a combined dry and
wet laboratory exercise, J. Chem. Educ. 91 (2014) 1017–1021.
[20] T.A. Hsu, G.T. Tsao, Convenient method for studying enzyme
kinetics, Biotechnol. Bioeng. 21 (1979) 2235–2246.
[21] T.A. Hsu, C.S. Gong, G.T. Tsao, Kinetic studies of
cellodextrins hydrolyses by exocellulase from Trichoderma
reesei, Biotechnol. Bioeng. 22 (1980) 2305–2320.
[22] A. Hernandez, M.T. Ruiz, An Excel template for calculation
of enzyme kinetic parameters by non-linear regression,
Bioinformatics 14 (1998) 227–228.
[23] M.V. Putz, A.M. Lacrama, V. Ostafe, Introducing logistic
enzyme kinetics, J. Optoelectron. Adv. Mater. 9 (2007)
2910–2916.
[24] B. Mannervik, Regression analysis, experimental error and
statistical criteria in the design and analysis of experiments
for discrimination between rival kinetic models, Methods
Enzymol. 87 (1982) 370–391.
[25] H. Akaike, A new look at the statistical model identification,
IEEE Trans. Automat. Contr. 19 (1974) 716–723.
[26] D.J. Birkett, R.A.J. Conyers, F.C. Neale, S. Posen, J.
Brudenell-Woods, Action of urea on human alkaline
phosphatases: with a description of some automated
techniques for the study of enzyme kinetics, Arch. Biochem.
Biophys. 121 (1967) 470–479.
[27] Z.E. Durak, M. Guru, Isolation, inhibition and kinetic
modelling of alkaline phosphatase (ALP) enzyme, J. Fac. Eng.
Archit. Gazi Univ. 28 (2013) 209–215.
[28] H.C. Hung, G.G. Chang, Multiple unfolding intermediates of
human placental alkaline phosphatase in equilibrium urea
denaturation, Biophys. J. 81 (2001) 3456–3471.