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

Enzyme inhibition studies by integrated

MichaelisMenten 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 Investigaco e de Tecnologias Agro-Ambientais e Biolgicas, CITAB, Universidade de Trs-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 MichaelisMenten 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

Keywords:

one inhibitor because reaction product is always present. This methodology is illustrated

integrated MichaelisMenten equation, assuming the simultaneous presence of more than

Integrated MichaelisMenten

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 tting by nonlinear

General mechanism kinetics

regression was performed with the Solver add-in (Microsoft Ofce 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.

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 reaction products accumulate. As previously explained there is
an incompatibility between the kinetic data and the underlying model [1]. To avoid this incompatibility the model must

be integrated to give a description of the reaction progress.

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 velocities is not theoretically accurate. Thus the kinetic studies
must evolve by the use of the integrated MichaelisMenten
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 MichaelisMenten equation considering 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

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The integrated MichaelisMenten equation in its classical implicit form [4,5] or in its explicit form [6] have other
advantages: (i) the initial addition of a product inhibitor can
be eliminated and Ki evaluated [710] allowing for example to
determine inhibition constants, of a particular reaction product isomer, more accurately since they are based on correct
stereochemistry and complete purity [11]; (ii) the possibility to
determine the inhibition constants without knowledge of true
substrate concentration [9,12,13]. This is of special mention in
heterogeneous reactions where the substrate is insoluble.
One of the main caveats regarding the use of integrated
MichaelisMenten equation to entire progress curve analysis is the possibility of gradual enzyme activity loss under
the assay conditions. However, nothing prevents its use with
much lower percent substrate depletion [2,5,10]. Thus, in this
work integrated equations will be applied only to data points
usually utilized to determine initial velocities (up to 10% substrate depletion).
The integrated MichaelisMenten equations have not been
usually applied in the presence of two inhibitors in the reaction medium. Nevertheless, integrated equations nd special
interest in studies with two different inhibitors since product inhibition is often found as a common regulating enzyme
mechanism [4,9,1417]. Thus, to study the inhibition kinetics
of this kind of enzymes it is necessary to consider the simultaneous presence of two inhibitors because one of them is a
reaction product (it is always present). The main objective of
this work is to develop and apply integrated equations to the
kinetic analysis of reactions carried out in the presence of two
inhibitors when one of them is a reaction product whether or
not mutually exclusive.

2.

Material and methods

2.1.

Enzyme reagents and reaction conditions

Alkaline phosphatase (E.C. 3.1.3.1.) was obtained from BDH.

All the other reagents used are readily available from
major suppliers such as Merck. Stock solutions of 4nitrophenylphosphate disodium salt and 4-nitrophenol were
prepared at concentrations of 20 mM. Reactions were carried out at 37 C containing 9.5 g enzyme in 2.75 ml reaction
volume and different amounts of 4-nitrophenylphosphate
(between 18.4 and 1500.0 ) in 0.1 M Tris/HCl buffer, pH 9.0.
The amount of product (4-nitrophenol) formed at several time
intervals (1060 s) was determined by absorbance at 405 nm,
using a spectrophotometer (path length, 1 cm) in conditions
that never surpass 10% of substrate depletion. Another set of
similar reaction experiments was also performed but in the
presence of 1.25 M urea (inhibitor).

2.2.

Data processing and analysis

Data processing and analysis was carried out by integrated

MichaelisMenten equations according to [5] but taking into
account the simultaneous presence of two inhibitors being
one of them a reaction product (Supplement 1 and 2). Diagnosis of enzyme inhibition and kinetic constants were estimated
by non-linear regression with Solver (Solver is an add-in

program included in the Excel software that is only necessary

to be activated to use them [5]) and discrimination between
different models was performed by Akaike information criterion (AIC) as previously explained [5,18]. Kinetic constants
were also estimated by initial velocities [19]. Both kinetic
methodologies used the same experimental data points.
Kinetic parameter uncertainties were determined by an Excel
template [22] and also by the software SPSS (Statistical Package
for the Social Sciences).

3.

Theory

Kinetic analysis with the integrated MichaelisMenten equations permits the determination of inhibition constants (Ki )
when more than one reaction product inhibits the enzyme
[9,14,16]. In this situation:
n

1
j=1

Kij

1
Ki

where Kij is the inhibition constant for one single product and
Ki is the global inhibition constant taking into account all products. In enzyme reactions characterized by the presence of two
different product inhibitors, Hsu and Tsao [20] showed that if
there is no addition of an initial inhibitor (at time zero) the
two constants, Kij1 and Kij2 cannot be individually determined
because they are in a lumped form. Nevertheless in the present
study a different condition occurs since urea is not a reaction
product which means that inhibition constants for urea and
phosphate are not lumped [17]. Furthermore, Orsi and Tipton
[4] advocated that when two inhibitors exhibit different inhibition types, linearization of integrated equations (e.g. [14]) is
not appropriate because different kinetic effect of inhibitors is
indistinctive. The use of non-linear regression is a simple way
to overcome linearization weakness [9].
All common types of linear inhibitions are included in
linear mixed inhibition model (MI) since some inhibition
constants can tend to innity, meaning that they are irrelevant
giving rise to other linear models (see Supplement 1) [5,10].
Assuming the mixed linear inhibition (MI) the following
MichaelisMenten rate equation is obtained:
v=

Km 1 +

Vmax [S0 ]
[I]
Kic

+ [S] 1 +

[I]
Kiu

(1)

where Km , Michaelis constant; Kic , inhibitor dissociation constant of enzyme-inhibitor complex; Kiu , inhibitor dissociation
constant of enzyme-substrate-inhibitor complex; Vmax , maximum velocity of reaction; v, initial velocity of reaction; [I]
concentration of inhibitor and [S0 ] initial concentration of substrate.
Considering the presence of two different inhibitors I1 and
I2 as well as different inhibition constants Kic1 and Kic2 or Kiu1
and Kiu2 the rate equation becomes:
v=

Km 1 +

Vmax [S0 ]
[I1 ]
Kic1

[I2 ]
Kic2

+ [S] 1 +

[I1 ]
Kiu1

[I2 ]
Kiu2

(2)

Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated MichaelisMenten equation considering 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

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Now assuming that inhibitor I1 is also formed during the

reaction ([I1 ] = [Pt ] + [I1,0 ] where [S0 ] [St ] = [Pt ]) and [St ] is the
concentration of substrate at the time t and the subscript 0
as S0 , I1,0 , means initial concentration of the I1 at t = 0, the rate
equation obtained is:
v=

d[S]
dt

Vmax [S0 ]

Km 1 +

[I1,0 ]
Kic1

[Pt ]
Kic1

[I2 ]
Kic2

+ [S] 1 +

[I1,0 ]
[Pt ]
Kiu1 + Kiu1

[I2 ]
Kiu2

 (3)

In Eq. (3), inhibition constants Kic1 and Kic2 or Kiu1 and Kiu2
are in a lumped form but after integration assuming that Pt
increases with time, the inhibition constants cease to be in a
lumped form [9,21].
Rearranging and integrating, gives:

Km 1 +

[I1,0 ]
Kic1

+[S0 ] 1 +

[St ]

dt =

[S0 ][St ]
Kic1

[I1,0 ]
Kiu1

[I2 ]
Kic2

[S0 ][St ]
Kiu1

[I2 ]
Kiu2


d[St ]

Vmax [S0 ]

[S0 ]

(4)

Or

dt Vmax = Km 1 +
0




[S0 ]
[I1,0 ]
[I2 ]
+
+
Kic1
Kic1
Kic2

Km
[S0 ]
[I1,0 ]
[I2 ]
1
+
+
+
Kiu1
Kiu1
Kiu1
Kiu2

1
Kiu1







[St ]

[S0 ]

1
d[St ]
[St ]

[St ]

d[St ]
[S0 ]

[St ]

[S]d[St ]

(5)

[S0 ]

Carrying out the integration (assuming Vmax = [E]kv where

[E] is the concentration of the enzyme and kv is a velocity
constant) the Eq. (6) is obtained.

t=

1
[E]kv

+ 1
+

1
2



Km 1 +

[S0 ]
[I1,0 ]
[I2 ]
+
+
Kic1
Kic1
Kic2

Km
[S0 ]
[I1,0 ]
[I2 ]
+
+
+
Kiu1
Kiu1
Kiu1
Kiu2
1
Kiu1


ln

[St ]
[S0 ]

([St ] [S0 ])

([St ]2 [S0 ]2 )

(6)

The Eq. (6) can be simplied, giving rise to simplied nested

models (Supplement 2) with fewer number of parameters such
as the uncompetitive inhibition (UC), noncompetitive inhibition (NCI), competitive inhibition (CI) or without inhibition
(WI) [5,10]. These models are simplications of model MI,
assuming that different constants (Kiu1 , Kic1 , Kiu2 , Kic2 ) tend to
innity. In Supplement 1 the equations are also rewritten in
order to be used in Excel software accommodating one or two
inhibitors or even no inhibitor. These equations have a wider
range of applicability than those used before [5] and should be
preferred.
These equations are always equations for predicting the
reaction time it takes to obtain a certain amount of product. The explicit form (expressed in terms of the W-Lambert

Table 1 Sum of square errors (SSE) values for different

models.

SSE
p
n

WI

CI

NCI

UCI

3976.3
2
112

1455.2
4
112

468.7
4
112

1019.0
4
112

MI
385.1
6
112

function) has the advantage to preserve statistical rigor since

substrate concentration is a function of time [6]. Due to tting
difculties 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
presented in this paper can be adapted to accommodate an
explicit form.

4.

Results and discussion

The utilization of Microsoft Ofce Excel software and

the Solver add-in to determine kinetic parameters of the
MichaelisMenten equation has been previously explained
[5,19]. The only difference is that the equations are more complex and there are two different concentrations of inhibitors.
To facilitate the insertion of equations in the appropriate Excel
cell, they were partitioned into , and  components as
explained in Supplement 1 and 2. Briey, it is necessary to ll
a blank worksheet with data points similarly to Excel spreadsheet (Supplement 2) and to calculate the time necessary to
obtain the product P (calculated time).
This kinetic investigation consisted of two parts: discrimination between available models (Tables 1 and 2) and
parameter estimation (Table 3). According to Table 1, models CI, NCI and UCI have the same number of parameters
and so it is enough to compare the SSE value for discrimination. The comparison reveals that NCI is better than any
other model since the SSE value is lower. However, the lowest
SSE value was obtained with the MI model, but the increase in
parameter numbers rise the incertitude about the best model.
Thus, statistical discrimination is necessary to compare NCI
with MI models. There are several methods to compare and
discriminate models such as extra sum-of-square F test, as
Mannervik [24] or Akaike information criterion (AIC) [25]. The
AIC methodology (Table 2) was chosen because it has the
advantage that kinetic models can be nested or non-nested
and the results give a continuous scale for model plausibility
as previously discussed [5,18]. The results show that the better model is the MI model with a probability near 100% and
should be preferred. For the same reason it is also necessary
to compare the WI with MI (Table 2) in order to rule out that
model WI is not appropriated. Experimental data points and
simulation lines using model MI with Solver optimized parameters (Table 3) are shown in Fig. 1A. To compare using initial
velocities methodology [19] the values are presented in Fig. 1B
and Table 3. Vmax (mol min1 mg1 ) can be obtained from kv
(M s1 (g/2.75 ml)1 ) [5]. The residual plots (Fig. 2) obtained
from data in Fig. 1 show the absence of systematic errors
once the residuals are scattered above and below of predicted
curve.
By the analysis of Table 3 it is evident than Kiu1 (uncompetitive inhibition constant of phosphate) tend to innity (is

Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated MichaelisMenten equation considering 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

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Table 2 Summary of model discrimination using Akaike information criterion methodology in the presence of
phosphate and urea.
Models A/B

SSEA

SSEB

WI/MI
NCI/MI

3976.3
468.7

385.1
385.1

112
112

PA +1

PB +1

AICA

AICB

AICcA

AICcB

Probability B correct

3
5

7
7

405.8
170.3

152.3
152.3

406.0
170.9

153.4
153.4

252.6
17.5

1.0
0.99984

Table 3 Summary of obtained constants with the best model (MI model).
MichaelisMenten

Km (M)

Kic1 (M)

Integrated equation
Initial velocities

41.4 1.2
58.3 2.1

7.0 1.5

Kiu1 (M)

Kic2 (M)

Kiu2 (M)

Vmax (mol min1 mg1 )

3.3 106 0.0

2.4 0.3
3.4 0.2

3.3 0.3
3.4 0.2

3.0 0.0
3.0 0.0

60

40

Residuals

Time (s)

50

30
20

15

30

45

60

-3

10
0
0

10

-6

Phosphate (M)

0.12

0.06
Residuals

mol min-1 mg-1

0
0

300

600

900

1200

1500

Time (s)

0.5

1.5

2.5

-0.06

4-Nitrophenyl phosphate (M)

Fig. 1 (A) Experimental data points and simulation lines

using model MI (integrated MichaelisMenten equation)
with optimized constants. Squares and continuous line
were obtained without inhibitor urea and circles and dash
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
in the presence of 1.25 mM urea.

-0.12

v (mol min-1 mg-1)

Fig. 2 (A) Residual plot from data of Fig. 1A. (B) Residual
plot from data of Fig. 1B. The plot patterns emphasize the
absence of systematic errors.

Table 4 Sum of square errors (SSE) values for different

models assuming only the assays in the absence of urea.
WI

too large compared to Kic1 ) and therefore phosphate (inhibitor

1) is predominately a competitive inhibitor. Urea (inhibitor 2)
presents similar values for Kiu2 and Kic2 , showing the predominance of non-competitive inhibition. If both inhibitors are of
the same type, for example competitive, it will be expected
that the best model is the competitive type. In this case the
discrimination of kinetic models was nished and the parameters can be obtained from the best model discriminated. The
results of Table 3 do not t in this situation because phosphate has a predominantly competitive inhibitor behavior and

SSE
p
n

1327.5
2
62

CI
83.1
3
62

NCI

UCI

120.2
3
62

114.8
3
62

MI
83.1
4
62

urea has a predominantly noncompetitive inhibitor behavior

(or eventually mixed).
Supplement 3 contains a summary of hypothetical kinetic
inhibition combinations obtained from possible results of the
best model discriminated. In the presence of different types
of inhibition (as in this study), it is necessary to conrm inhibition patterns of phosphate and urea individually.

Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated MichaelisMenten equation considering 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

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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

WI/CI
CI/MI

1327.5
83.1

83.1
83.1

n
62
62

PA +1

PB +1

AICA

AICB

AICcA

AICcB

3
4

4
5

196.0
26.1

26.1
28.1

1964.4
26.8

26.8
29.2

Table 6 Modications in the MI model to evaluate the

inhibition pattern of the second (urea) inhibitor (see
text).
MI model
modied

Changes to make
in the MI model

Number of
parameters
remaining

WIm

Removing [I2 ]/Kic2

(G10/$B$3) and [I2 ]/Kiu2
(G10/$B$6)
Removing [I2 ]/Kiu2
(G10/$B$6)
Changing [I2 ]/Kiu2
(G10/$B$6) to [I2 ]/Kic2
(G10/$B$3)
Removing [I2 ]/Kic2
(G10/$B$3)
Do not remove anything

CIm
NCIm

UCIm
MI

3
3

3
4

Table 7 Sum of square errors (SSE) values for different

models assuming the modications of Table 6 (in the
presence of phosphate and urea).

SSE
p
n

WIm

CIm

NCIm

UCIm

4258.3
2
112

1456.0
3
112

402.3
3
112

1096.9
3
112

MIm
392.6
4
112

In this case to say categorically that the phosphate is a

competitive inhibitor it is necessary to analyze the results
considering only assays with phosphate (initially present or
not) as explained previously [10]. In this study there is not
phosphate initially present but only generated as a reaction
product that increases with time. To do this is necessary to
analyze the experimental points obtained without the presence of urea (in Excel template are only considered the values
listed until the line 72) although used equations are the same.
In this situation when nonlinear regression is performed, the
quotients G10/$B$3 and G10/$B$6 have no inuence on the
equation because cell G10 is always zero (cells $B$3 (Kic2 ) and
$B$6 (Kiu2 ), do not change because they do not interfere in the
equation). The results obtained in this situation are showed
in Tables 4 and 5 and it is possible to conclude that CI is better than MI with a probability of 77%, and also better than WI
with a probability of 100%. The inhibition constant obtained
was Kic1 = 10.7 M.
In order to evaluate the inhibition pattern of urea, only
a modied MI equation is used. As phosphate is a reaction


169.6
2.4

Probability B correct
1.00
0.23

product it is not possible to study the inhibition of urea without the presence of phosphate. Thus, it is necessary to copy
MI equation model (Supplement 1) to ve spreadsheets and
to give e.g. the following names to the ve spreadsheets
(WIm , CIm , NCIm , UCIm and MIm ; when m means modied).
Modications of MI model are necessary because phosphate
inhibition constants (Kic1 and Kiu1 ) are now xed values and
it is not possible to do that with the equations of Supplement 1. Thus Kic1 (cell B2) and Kiu1 (cell B5) will be xed by
inserting in each spreadsheet the values obtained in previous study without the presence of urea (Kic1 = 10.7 M and
Kiu1 = (e.g. 3.3 106 M obtained in Table 3), i.e. converting
them in non-variable parameters). Additionally, other minor
changes in each of the ve spreadsheets should be performed
as explained in Table 6. Before carried out nonlinear regression, do not forget to remove in the option by change the
cell (Solver dialog box) the cells $B$2 (Kic1 ) and $B$5 (Kiu1 ).
The discrimination result in Tables 7 and 8 show that urea
is a mixed inhibitor with a probability of 57%. The constants
obtained were Kic2 = 2.6 M and Kiu2 = 3.3 M.
Data points and model MI simulation with solver optimized constants (Table 3, integrated equation) are presented
in Fig. 1A. The constants obtained with the integrated
MichaelisMenten equation and by traditional methods (initial velocities) are compared in Table 3. It should be pointed
out that determination of inhibition constants, Kic1 and Kiu1 ,
by the MichaelisMenten equation (initial velocities) requires
the initial addition of phosphate in another set of assays
without urea. As above explained, the study of urea inhibition alone by initial velocities is not theoretically possible
because phosphate is a reaction product. Several authors concluded that urea is a noncompetitive inhibitor of alkaline
phosphatase [26,27]. Nevertheless, in this work, the probability of alkaline phosphatase inhibition by urea to be of the
noncompetitive type is not fully supported because slightly
different inhibition constants (Kic2 and Kiu2 ) were obtained
in the MI model discriminated (Table 8) by Akaike information criterion. This difference could be due to the fact that
effect of phosphate inhibition was not considered in traditional methods by initial velocities. Now it is possible to
state that phosphate is a competitive inhibitor and urea is
a mixed inhibitor with the inhibition constants presented in
Table 3.
It is important to point out that some alkaline phosphatase
isoenzymes present a reversible inhibition by urea (up to 2 M)

Table 8 Summary of model discrimination using Akaike information criterion methodology assuming the
modications of Table 6 (in the presence of phosphate and urea).
Models A/B

SSEA

SSEB

NCIm /MIm

402.3

392.6

112

PA +1

PB +1

AICA

AICB

AICcA

AICcB

150.5

148.4

151.6

151.1

0.54

Probability B correct
0.57

The subscript m in NCIm /MIm denotes that are modied MI equations as explained in text.
Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated MichaelisMenten equation considering simultaneous presence of two inhibitors when one of them is a reaction product, Comput. Methods Programs Biomed. (2015),
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and others are more sensitive and present an irreversible inhibition by denaturation [26,28], a phenomenon not observed
in this study. The use of the integrated MichaelisMenten
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 MichaelisMenten

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 initial velocities. The possibility of utilizing this methodology to
determine kinetic constants in general kinetic studies and the
possibility of developing spectrophotometer software to indicate on line and in real time the type of inhibition and kinetic
constants is important to point out.

Conict of interest
None.

Acknowledgment
Authors are grateful for the nancial 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.

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Please cite this article in press as: R.M.F. Bezerra, et al., Enzyme inhibition studies by integrated MichaelisMenten equation considering 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