Analytical Biochemistry 572 (2019) 58–62

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

A reanalysis of protein tyrosine phosphatases inhibitory studies using the T

unnatural substrate analogue p-nitrophenyl phosphate
Ryan Walsh
Department of Microbiology and Biochemistry, INRS–Institut Armand-Frappier, Laval, Québec, H7V 1B7, Canada

ARTICLE INFO ABSTRACT

Keywords: The determination of inhibition mode is extremely important in the understanding of drug interactions and
Enzyme inhibition biological mechanisms. The data presented by Hjortness et al. in their recent papers [1,2] on the inhibition of
Global data fitting various Protein Tyrosine Phosphatases addresses this issue in an exemplary manner, determining the mode of
inhibition based on global fitting of the data to multiple models of inhibition. However, Protein Tyrosine
Phosphatases are known to undergo substrate induced conformational changes, so inhibition models which are
based on enzyme that adhere to Michaelis-Menten single substrate kinetics may not be appropriate for ex-
amining these interactions. To examine the appropriateness of these models, the reported raw data was ex-
amined using a recently developed template for global data fitting in Excel. Based on the sum of squared re-
siduals this analysis demonstrates that the excel template was able to match or improve on the reported fittings
and demonstrates that a better fit can be achieved with a model that takes into account p-nitrophenyl phosphate-
based substrate activation. Whether the substrate activation observed with this model substrate has physiolo-
gical relevance is debatable, however, it does correspond to the known conformational rearrangement these
enzymes undergo when working on their larger peptide substrates.

Enzyme inhibition modeling has long relied on the interpretation of
plotted data where pattern recognition has been the predominant
method for inhibitor classification. With the advent of computer-based
data analysis, global data fitting can be used to both compare and
identify modes of inhibition in a less biased way. This approach is
utilized by Hjortness et al. [1,2], to examine the inhibition of several
Protein Tyrosine Phosphatases with classical total inhibition models:
competitive, non-competitive, uncompetitive and mixed non-competi-
tive inhibition. This sort of analysis is somewhat rare but has been re-
ferred to as a technically impeccable method of determining inhibition
mode [3]. While the fitting was not published the raw data, sum of
squared residuals (RSS) for the fits and criteria for identifying inhibition
modes based on F-tests and Akaike's information criterion (AIC) [4]
were disclosed [1,2]. Specifically the studies stated AIC values were
used to compare single parameter models while F-tests were used to
compare the single parameter models to the more complex 2 parameter
mixed non-competitive equation. However, AIC values are already ne-
gatively scored based on increasing model complexity and as such could
have been used to compare the mixed non-competitive model with the
others. The publication of the experimental data associated with the
inhibition experiments allowed for the reanalysis of the fits using a
template developed in excel for global fitting of enzyme inhibition and
activation data [5]. Based on the previously reported RSS values,
produced by fitting their data to the competitive, non-competitive,
uncompetitive and mixed non-competitive inhibition models, the excel
based template was able to match or improve on the fittings in all but
one example, the inhibition of PTP1B with IA (Table 1). In addition, the
modifier equation (Eq (1)) was able to match or improve on the best fits
produced by the other inhibition models in all the datasets. The
modifier equation can be regarded as non-mechanistic as it simply
describes the presence of two catalytically relevant enzyme species in
solution one acting on substrate alone and another affected by the in-
hibitor or activator.
[S ] [X ]
v= VS1 (VS1 VS 2 )
(
[S ] + K S1 (K S1
[X ]
K S 2) [X ] + k
Sx ) [X ] + kSx
(1)
However, Protein Tyrosine Phosphatases are known to have com-
plex substrate interactions where substrate binding produces a re-
arrange in their conformation [6,7]. An analysis of the uninhibited
enzymes suggested that PTP1B and SHP2 both exhibited marked sub-
strate modulation while TC-PTP did not (Table 2; Fig. 1). This suggested
that a more appropriate model for the inhibition would include sub-
strate modulation [8,9]. The substrate modulation equation (shown in
Table 2) works in a similar fashion to the modifier equation dividing the

E-mail address: Ryan.Walsh@iaf.inrs.ca.

https://doi.org/10.1016/j.ab.2019.02.025
Received 25 January 2019; Received in revised form 21 February 2019; Accepted 25 February 2019
Available online 04 March 2019
0003-2697/ © 2019 Elsevier Inc. All rights reserved.
R. Walsh Analytical Biochemistry 572 (2019) 58–62

Table 1
Sum of squared residuals and Δ Akaike's information criterion values for the global data fitting to inhibition models.

Comparison of inhibition models for the inhibition of protein tyrosine phosphatases using the sum of squared residuals from global data fitting. Values reported in the
gray boxes represent previously published fits with (*) denoting the determined mode of inhibition [1,2]. The other values represent the sum of squared residuals
produced with the excel template [5] which includes the modifier equation (Eq. (1)) the modifier equation containing Hill coefficients in the inhibitor binding term
(Eq. (3)) and fitting of the data to the more complex substrate modulated equations (Eqs. (2) and (4)). The (†) and green boxes denote the model which produced the
best fit based on the AIC, while the red boxes highlight the minimum sum of squared residuals.

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R. Walsh Analytical Biochemistry 572 (2019) 58–62

Table 2
Sum of squared residuals and kinetic constants for the global data fitting of the Michaelis-Menten and substrate activation equation.
Michaelis-Menten equation Substrate Modulation
[S] [S ] [S ] [S ]
v= V v= VS1 VS1 + VSS1
[S] + K s1 S1 [S] + K S1 [S] + K SS1 [S] + K SS1

KS1 VS1* RSS ΔAIC KS1 KSS1 VS1* VSS1* VSs1 ** RSS ΔAIC
μM μM/s μM μM μM/s μM/s VS1

PTP1B 276 0.81 0.0660 14.00 188 19 415 0.73 1.04 1.42 0.0237 0.00
TC-PTP 200 0.84 0.0351 0.00 200 200 0.71 0.84 1.18 0.0351 3.99
SHP2 1420 0.23 0.0924 30.52 93 10 232 0.08 0.32 4.25 0.003 0.00

Comparison of kinetic models for protein tyrosine phosphatases using ΔAIC and sum of squared residuals values obtained from global data fitting. Substrate affinity
values represent the global minimums, while *the rates represent the average of the best fit values produced by the individual experimental datasets. **For the
substrate activation model, the rates were similarly maximized for each dataset, however, the ratio of substrate activation was fit globally to all the datasets.

Fig. 1. Comparison of the fit to the A) Michaelis-Menten equation and the B) substrate activation equation for PTP1B (blue), TC-PTP (green) and SHP2 (yellow). (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

catalytically relevant enzyme species into two populations, one with a v=

[S ]
VS1 (VS1 VS 2)
[X ]H 1
secondary substrate interaction and one without. This segregation of [S] + K S1 (K S1 K S2) H 1
[X ]H 1 [X ]H1 + kSxH1

populations allows the addition of modifier terms (Eq (2)) akin to those [X ] H1
+ kSx

found in the modifier equation (Eq (1)). [S ]

VS1 (VS1 VS 2)
[X ]H 1
[X ]H 2 [X ]H 1 + kSx H 1
[S] + K SS1 (K SS1 K SS 2) H 2 H1
[S ] [X ] [X ] + kSSx
v= VS1 (VS1 VS 2)
[X ] [X ] + kSx [S ] [X ]H 2
[S] + K S1 (K S1 K S2) + VSS1 (VSS1 VSS 2)
[X ] + kSx
[X ]H 2 [X ]H 2 + kSSx
H1
[S ] [X ] [S] + K SS1 (K SS1 K SS 2) H 2
VS1 (VS1 VS 2) [X ] H1
+ kSSx (4)
[X ] [X ] + kSx
[S] + K SS1 (K SS1 K SS 2)
[X ] + kSSx

[S ] [X ] Additionally, the template was modified to include AIC calculations

+ VSS1 (VSS1 VSS 2)
[S] + K SS1 (K SS1 K SS 2)
[X ] [X ] + kSSx and the boxplot of residuals was expanded to include the additional
[X ] + kSSx (2) equations. Based solely on the sum of square residuals, Eq (4) produced
a better fit than all the other models with every data set (Table 1 red
As the excel template does not include an equation that considers
boxes). However, evaluation based on the AIC values suggested that in
substrate modulation an additional sheet was added so the results could
all but five of the data sets Eq (4) may represent an overfitting of the
be compared to the other inhibition models. While the substrate mod-
data (Table 1, green boxes). For instance, in the case of PTP1B inhibi-
ulation equation did provide an improvement to the fitting of some of
tion by CA, while Eq (4) does produce the best fit, the improvement
the inhibition data in some instances the inhibition patterns produced
over the fit produced by the mixed non-competitive equation is not very
by some of the data sets suggested varying the Hill coefficient could
significant. This is reflected in the ΔAIC value where Eq (4) is calculated
potentially provide an additional improvement in the fit. Therefore
as 7.59 in contrast with the mixed non-competitive equation that pro-
equations (1) and (2) were modified to include hill coefficients in the
duces the best fit to this data. In general, models should fall in the range
inhibitor binding terms (Eqs (3) and (4)).
of 0–3 of the best fit model for consideration as potential useful alter-
natives while those above 10 can be discounted. This is evident with the
[S ] [X ]H1
v= VS1 (VS1 VS 2 ) H1
AA inhibition of PTPB1 where Eq (4) produces the best fit according to
[X ]H 1 [X ]H 1 + kSx the RSS and AIC, but proximity of the AIC for the mixed non-compe-
[S ] + K S1 ( K S1 KS2)
[X ]H 1 + kSx
H1
titive equation (ΔAIC 0.06) suggests it may also provide a viable model
(3) of this interaction. However, the low ΔAIC scoring of the simpler
models in this analysis may result from a bias in the way the data was
collected, where all the reported inhibitor concentrations were kept the

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R. Walsh Analytical Biochemistry 572 (2019) 58–62

Fig. 2. Comparison of the fits between A) the reported mixed non-competitive inhibition of PTP1B by IA, with B) the modeling of the inhibition using eq. (2), C) the
reported mixed non-competitive inhibition of TC-PTP by CA, with D) the modeling of the inhibition using eq. (2), E) the reported non-competitive inhibition of SHP2
by DeAA, with F) the modeling of the inhibition using equation (2).

same. This would not be an issue if the inhibitors all had the same
binding affinity, however the consistent use of 100, 200, 300 and
400 μM resulted in random data collection along the inhibitor binding
curves. For example, the inhibition of PTPB1 and TC-PTP by DeAA
appears to skew towards less complex models as the inhibitor binding
constant for DEAA falls in the range of 1000 μM for both enzymes
(supplementary materials). This means the collected data only displays
a small portion of the inhibition profile. The opposite situation occurs
with BBR inhibition of PTPB1, where the inhibitor binding affinity
produced by the best fits for all the models falls in the range of 40 to
0.3 μM (supplementary materials) placing all the inhibitor concentra-
tions significantly above the binding constants. Additional skewing
toward the simpler models seems to result from noise, as is observable
with DeAA inhibition of TC-PTP, where the sum of squared residuals is
very similar across all the models (Table 1) and individual data points
for different inhibitor concentrations are observed to overlap (supple-
mentary materials). This overlap is also apparent in other datasets that
were examined (Fig. 2).

61
R. Walsh
Based on the RSS and AIC values it is clear that the more complex
models provide better fits (Table 1). It can also be stated that SHP2 and
PTP1B exist in multiple catalytic states induced by substrate con-
centration (Table 2). Where the PTPB1 and SHP2 enzymes produces a
clear improvement when substrate activation is considered the TC-PTP
does not, as the substrate binding constants for the two forms converge
without an improvement in RSS. As Such, using an equation that ac-
counts for four catalytically relevant states, the enzyme working on one
substrate molecule, the enzyme activated by a secondary substrate
molecule and both of these states being affected by inhibitor (Equation
(2) and (4)) provides a marked improvement in the fitting of the PTP1B
and SHP2 datasets (Table 1, Fig. 2). Additionally, the inclusion of Hill
coefficients in the inhibitor binding terms (Eqs. (2) and (4)), greatly
improved the overall filling of all the datasets (Table 1), providing
further evidence that conformational rearrangement is integral to the
catalytic activity of these enzymes [6,7].
However, the relatively small size of the p-nitrophenyl phosphate
substrate used in these studies may also indicate that this substrate has
difficulty producing the conformational changes in the protein tyrosine
phosphatases associated with the dephosphorylation of larger peptide
substrates [6,7]. This may indicate that the Protein Tyrosine Phospha-
tases are able to hydrolyze this smaller substrate analogue without
undergoing the conformational rearrangement associated with their
larger peptide substrates. As such, whether the apparent substrate ac-
tivation suggested by this data has physiological relevance remains to
be determined and should be examined carefully as the use of p-ni-
trophenyl phosphate in such studies has led to mechanistic ambiguity
before [10]. Nonetheless, the shift in substrate hydrolysis produced by
the p-nitrophenyl phosphate may provide more insight into the catalytic
activity of these enzymes.
Notes
The author declares no competing financial interests.
Funding sources
NA.
Abbreviations
AA abietic acid
62
Analytical Biochemistry 572 (2019) 58–62
BBR 3-(3,5-Dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-
sulfonicacid-(4-(thiazol-2-ylsulfamyl)-phenyl)-amide
CA continentalic acid
DeAA dehydroabietic acid
DiAA dihydroabietic acid
IA isopimaric acid
PTP1B Protein tyrosine phosphatase 1B (accession ID: P18031)
TC-PTP T-cell protein tyrosine phosphatase (accession ID: P17706)
SHP2 protein tyrosine phosphatase nonreceptor type 11 (accession
ID: Q06124)
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ab.2019.02.025.
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