Biochimie 176 (2020) 12e20

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Biochimie
journal homepage: www.elsevier.com/locate/biochi

Relationship between enzyme concentration and Michaelis constant

in enzyme assays
Kyong-Il Yun*, Tong-Sul Han
Department of Biophysics, Faculty of Life Science, KIM IL SUNG University, Taesong District, Pyongyang, Democratic People’s Republic of Korea

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

Article history: The upper bound of enzyme concentration for accurately estimating the parameters in Michaelis-Menten
Received 25 February 2020 (MM) equation is not completely determined and still under discussion, even though many researchers
Received in revised form have investigated the equation’s validity for a long time. In the paper, we broadly investigated the
7 June 2020
correlation between the system of ordinary differential equations for monosubstrate irreversible enzyme
Accepted 9 June 2020
reaction (HMM-system) and its derivative MM equation focusing on the relationship between initial
Available online 23 June 2020
enzyme concentration [E]0 and Michaelis constant Km by numerical simulation. According to the results,
the initial reaction velocity v0 is still a function of initial substrate concentration [S]0 at [E]0<Km. The
Keywords:
Michaelis-Menten (MM) equation
function is identical to the MM equation at [E]0&0.01Km, while it is described as a new type of equation
Michaelis constant at 0.01Km&[E]0<Km. This function is of great significance in enzyme assays as a comprehensive
Enzyme concentration approximation for the HMM-system.
Enzyme assay © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights
Parameter estimation reserved.
Numerical simulation

1. Introduction characteristics at a certain condition. These parameters are esti-

Michaelis-Menten (MM) equation [1e3], a fundamental equa-
tion of enzyme kinetics, has been widely used in biology for the last
one century [4]. Surprisingly, theoretical investigation on the val-
idity of MM equation is not completed and still under discussion
[5]. Fundamental reason is that the system of ordinary differential
equations of monosubstrate irreversible enzyme reaction (HMM-
system [6]) has no analytical solution [7]. Up to now, there are
different kinds of assumptions applied to approximate the dy-
namics of HMM-system, such as the quasi-steady-state assumption
(QSSA) [3,8,9], the total quasi-steady-state assumption (tQSSA)
[10,11], and the reactant stationary assumption (RSA) [12]. That is
why there are several approximations such as the MM equation, the
reverse Michaelis-Menten (RMM) equation [13,14], the equilibrium
chemistry approximation (ECA) model [15e17], and the PEA model
[18], and so on.
Meanwhile, experimentalists have used the MM equation in
characterizing the enzymes from the beginning [19e21]. The MM
equation contains the parameters: Michaelis constant Km and
maximum reaction velocity Vmax that reflect the enzyme
* Corresponding author.
E-mail address: ki.yun0605@ryongnamsan.edu.kp (K.-I. Yun).
mated through two types of experiments: initial rate experiment
[22] and time-course experiment [23], but the former is more
commonly used as it has the advantages of reducing possible in-
terferences from reversible reaction, enzyme instability, and
product inhibition and thus of being applied to much more
complicated reactions [24].
In the initial rate experiments, the MM equation is, most likely,
considered as the relationship between initial substrate concen-
tration [S]0 and initial reaction velocity v0 [25], but such an adop-
tion should be based on the QSSA and the RSA [5]. The criterion for
validity of the RSA is more stringent than that required for the
QSSA, but itself is not a sufficient condition for enzyme assays.
In enzyme assays, there are some ordinary ranges for reaction
conditions including temperature, buffer concentration and pH,
and substrate concentration, but as for enzyme concentration,
there is no existence of such a range and it is experimentally
determined to measure initial velocity [22]. Whenever enzyme
stability is very low, or reaction progression is very slow, it would
be favorable to raise enzyme concentration higher to improve
measurement sensitivity. Furthermore, the development of stra-
tegies for studying fast enzyme kinetics in the resolution of mili-
seconds or even nanoseconds allows raising enzyme
concentrations much higher than usual measurement conditions
[25e27]. A question is whether it is possible to estimate the

https://doi.org/10.1016/j.biochi.2020.06.002
0300-9084/© 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
 K.-I. Yun, T.-S. Han / Biochimie 176 (2020) 12e20
characteristic parameters of an enzyme at high enzyme concen-
trations as long as initial reaction velocities are measurable.
Although many researchers have discussed about enzyme ki-
netics at high enzyme concentrations [11,20,28e32], the criterion
of whether enzyme concentration is high or low is indefinite,
especially in relation to enzyme assays. Often, the relationship
between [E]0 and [S]0 is highly topical in enzyme kinetics. For all
that, it is not reasonable to formulate kinetic laws uniformly by
relationship between [E]0 and [S]0, because kinetic behavior of
every enzyme must be distinguished by its characteristics so as to
meet the purpose of enzyme assay.
Therefore, it is necessary to investigate the upper bound of [E]0
for enzyme assays on the basis of enzyme characteristics. Michaelis
constant Km, as a function of the three rate constants, and with the
dimension molarity, must be a unique standard for the purpose.
In the paper, we broadly investigated the correlation between
the HMM-system and its derivative MM equation focusing on
relationship between [E]0 and Km, and paying special attention to
elucidate the upper bound of [E]0 for enzyme assays.
2. Methods
Often, one can describe the monosubstrate irreversible enzyme
reaction as the following.
k1
E þ S ƒƒƒƒƒƒƒƒƒƒ
ƒ! ES ƒƒƒƒƒƒƒƒ!
k2
) P þ E;
k1r
where E stands for enzyme, S for substrate, ES for enzyme-substrate
complex, and P for product, and then k1, k1r, and k2 are the rate
constants for the corresponding unitary reactions respectively.
Applying the law of mass action to the above reaction, one can
obtain the system of ordinary differential equations abbreviated as
HMM-system [6]. Under the QSSA [3,8,9] and the RSA [12], the
HMM-system can reduce to MM equation:
v0 ¼ Vmax ½S0 =ðKm þ ½S0 Þ; (1)
where v0 is initial reaction velocity, [S]0 initial substrate concen-
tration, Km Michaelis constant and Vmax maximum reaction velocity.
Km and Vmax are defined as the following relationships:
Vmax ¼ k2 ½E0 ; (2)
Km ¼ ðk1r þ k2Þ=k1; (3)
where [E]0 is initial or total enzyme concentration.
Now, let’s consider correlation between the HMM-system and
the MM equation.
For solving HMM-system, initial conditions and rate constants
were set as the followings.
Initial enzyme concentration [E]0 was set in the range of
(106e10 )Km with the step 10 .5Km. The ranges of initial substrate
concentration [S]0 were set around Km: (100.5e100.5)Km,
(101e101)Km, and (103e103)Km, with the steps of 10 .1Km, 10 .2Km,
and 10 .5Km, respectively, or set as [S]0¼(105e105)Km with the step
10 .5Km. Also, initial concentrations of enzyme-substrate complex
and product were set as [ES]0 ¼ 0 and [P]0 ¼ 0, respectively.
The rate constants k1, k1r, and k2 (¼kcat) were set to take the
values within the ranges of 101e107 mM1min1, 101e107 min1,
and 104e107 min1 with tenfold steps, respectively, taking into
consideration the known values of Km, kcat and the other rate
constants [7,33,34], and then final combinations were selected
within the range of Km ¼ 104e102mM as calculated from each
combination by Eq. (3). Eventually, the final combinations could
13
represent almost all kinds of enzymes with different rate constants.
Initial velocity v0 was calculated by Eq. (4):
v0 ¼ k2½ESmax ; (4)
where [ES]max is the concentration of ES complex at d [ES]/dt ¼ 0.
Strictly, v0 in Eq. (4) is the reaction velocity at steady-state and
occurs at very early phase, so it is a true or theoretical initial
velocity.
Reaction time was set as 1min, which was enough to record
[ES]max in every case.
The HMM-system was solved by using variable order method
based on the numerical differential formulas [35] to obtain dataset
of [S]0 to v0, from which the parameters Km and Vmax were esti-
mated by using linear least-squares method with the three types of
linear transformations: Hanes [36], Lineweaver-Burk [37], and
Eadie-Hofstee [38,39] plots, respectively. And the abilities of the
linear transformations to fit the curves of [S]0~v0 were evaluated
with correlation coefficient R2.
The parameter values determined by Eq. (2) and Eq. (3) with the
prescribed values of rate constants and initial enzyme concentra-
tions were described as “true values” and symbolized as “V”max and
“K”m respectively, while those estimated inversely from the HMM-
system simulation data were described as “estimates” and sym-
bolized as “Vmaxa” and “Kma” respectively. Similarily, initial velocity
calculated by Eq. (4) was described as “real velocity” and symbol-
ized as “v0”, while the corresponding estimate was described as
“estimated velocity” and symbolized as “ve”.
Estimation deviation was evaluated with absolute and/or rela-
tive deviation. The estimates with relative deviation below 10%
were adopted as the accurate.
All units were composed of mM and/or min, otherwise specified.
3. Results
3.1. One percent of Km is the upper limit of enzyme concentration
for simultaneous estimation of the parameters in MM equation
through initial rate experiment
Firstly, within the range of [E]0¼(106e10 )Km, we investigated
estimation deviation for Km and Vmax with respect to the ratios Km/
[E]0, the [S]0 ranges and the estimation plots, respectively (Fig. 1).
In Fig. 1A, D, and 1G, most of R2 values were very close to 1 at
almost all enzyme concentrations, regardless of the estimation
plots and the [S]0 ranges. At log10(Km/[E]0) ¼ 0, R2 values for some of
the enzymes with Km z 100 mM(Fig. 1A and D) or
kcat&1.1  103min1 (Fig. 1G) remarkably diminished, especially
in Eadie-Hofstee plot. Therefore, the hyperbolic equation for [S]0
and v0 is valid at [E]0<Km:
v0 ¼ Vmaxa ½S0 =ðKma þ ½S0 Þ: (5)
however, R2 z 1 do not assure Kma ¼ Km and Vmaxa ¼ Vmax in Eq. (5).
Indeed, the relative deviations for Km were dependent on the
ratios Km/[E]0, the [S]0 ranges, and the estimation plots (Fig. 1B, E,
1H). At all the [S]0 ranges and the estimation plots, the Km estimates
deviated remarkably with increasing [E]0 within log10(Km/[E]0)&2
but most narrowly within log10(Km/[E]0)¼(2e3) for all the enzymes,
whereas they deviated beyond 10% within log10(Km/[E]0)¼(4e6)
only for some enzymes different according to the estimation plots
and the [S]0 ranges (Table 1).
Likewise, the relative deviations for Vmax varied with the ratios
Km/[E]0, the [S]0 ranges and the estimation plots, respectively
(Fig. 1C, F, and 1I). At all the [S]0 ranges and estimation plots, the
Vmax deviations were narrowest within log10(Km/[E]0)¼(2e3). They
14 K.-I. Yun, T.-S. Han / Biochimie 176 (2020) 12e20

Fig. 1. Estimation accuracies for Km and Vmax mainly depend on the ratios of Km to [E]0. In each subgraphs, colors represent the estimation plots, blue: Hanes, red: Lineweaver-
Burk, green: Eadie-Hofstee. The ranges of [S]0 are (100.5e100.5)Km in A, B, C; (101e101)Km in D, E, F; (103e103)Km in G, H, I, respectively. In H and I, the estimation results by
Lineweaver-Burk plot were not shown because their deviations were much broader than those by the other plots, and, in particular, many of the estimates were negative within
log10(Km/[E]0)<3.

also increased with increasing [E]0 within log10(Km/[E]0)&2 but
their deviation degrees were much smaller than those for Km and
decreased significantly with increasing the range of [S]0. Again, the
enzymes with estimation deviation beyond 10% but less than 20%
for Vmax were almost identical at each [S]0 ranges and estimation
plots (excluding Lineweaver-Burk plot at [S]0¼(103e103)Km)
within log10(Km/[E]0)¼(4e6) (Table 2).
As shown in Fig. 1, Tables 1 and 2, Lineweaver-Burk plot always
resulted in wonderful R2 but the poorest estimates at all the [S]0
ranges, while the deviation degrees rather increased with
increasing the range of [S]0 in contrast to Hanes or Eadie-Hofstee
plot. Especially at the range [S]0¼(103e103)Km, Lineweaver-Burk
plot resulted in much broadly deviated estimates at all the ratios
of Km/[E]0, even negative Kma and Vmaxa within log10(Km/[E]0)<3, so
the results were not shown in Fig. 1H and I. Eadie-Hofstee plot
generated the smallest R2 (still greater than 0.99 in most cases) but
the estimation results were comparable to those of Hanes plot.
We investigated the ranges of Km/[E]0 for accurately estimating
Km or Vmax for all the enzymes at each [S]0 ranges.
As shown in Table 3, with increasing the range of [S]0, the upper
 K.-I. Yun, T.-S. Han / Biochimie 176 (2020) 12e20 15

Table 1
Enzymes with estimation deviation beyond 10% for Km at log10(Km/[E]0)¼(4e6).

log10 ([S]0 /Km) Plot type Enzyme kinds Maximum deviation (%)

log10 (k1) log10 (k1r) log10 (k2) Km (mM)

0.5e0.5 Hanes 5e6 1e2 4e2 &1 11.9

Lineweaver-Burk 4e7 1e5 4e5 &11 11.9
Eadie-Hofstee 4e7 1e5 4e5 &11 11.9
1e1 Hanes 3e6 1e2 4e2 &11 22.8
Lineweaver-Burk 3e7 1e6 4e6 &110 13.3
Eadie-Hofstee 4e7 1e5 4e5 &11 12.3
3e3 Hanes 4e5 1 4e1 &2 36.7
Lineweaver-Burk 1e7 1e7 4e7 &100a 67.0
Eadie-Hofstee 4e7 1e4 4e4 &200 16.3
a
Unit: mM.

Table 2
Enzymes with estimation deviation beyond 10% for Vmax at log10(Km/[E]0)¼(4e6).

log10 ([S]0 /Km) Plot type Enzyme kinds Maximum deviation (%)

log10 (k1) log10 (k1r) log10 (k2) Km (mM)

0.5e0.5 Hanes 5e7 1e4 4e4 &2 18.5

Lineweaver-Burk 4e7 1e5 4e4 &10 18.5
Eadie-Hofstee 5e7 1e4 4e4 &2 18.5
1e1 Hanes 4e7 1e4 4e4 &2 18.5
Lineweaver-Burk 4e7 1e4 4e4 &11 18.5
Eadie-Hofstee 5e7 1e4 4e4 &2 18.5
3e3 Hanes 4e7 1e4 4e4 &2 17.3
Lineweaver-Burk 1e7 1e7 4e7 &100a 64.1
Eadie-Hofstee 4e7 1e4 4e4 &2 17.7
a
Unit: mM.

bound of [E]0 for accurately estimating Vmax increased whereas
those for Km changed little. The estimation deviations of Km and/or
Vmax for some enzymes were beyond 10% at log10(Km/[E]0)S4,
regardless of the [S]0 ranges and the estimation plots. The ranges of
Km/[E]0 for accurate estimation were different at each estimation
plots and [S]0 ranges. Surely, the Hanes plot was the best. The
ranges of Km/[E]0 for parameter estimation by Lineweaver-Burk plot
decreased with increasing the range of [S]0 and completely dis-
appeared at the range [S]0¼(103e103)Km. The range
[S]0¼(100.5e100.5)Km is worthy notice, because the ranges of Km/
[E]0 for parameter estimation were nearly identical and broadest at
each estimation plots.
Assuming that typical [S]0 range be within the tenfold higher
and lower concentrations around Km, the upper bound of [E]0 for
estimating Km and Vmax simultaneously would be at log10(Km/
[E]0) ¼ 2: that is one percent of Km, which was still valid even at the
range [S]0¼(103e103)Km in case of Hanes or Eadie-Hofstee plot
(Fig. 1H and I, Table 3).
3.2. Estimation deviation for Km at the enzyme concentrations
beyond 1% of Km carries information on enzyme concentration and
relationship between k1r and k2
In Fig. 1, the estimation deviations for Km increased with
increasing [E]0 towards Km at each estimation plots for all the [S]0
ranges, whereas those for Vmax decreased with increasing the range
of [S]0, implying that the former be significantly dependent on [E]0
rather than the latter.
Therefore, we investigated relationship between absolute de-
viation of Km estimate and initial enzyme concentration [E]0 within
the range of [E]0¼(102e100.5)Km, where [E]0 ¼ Km was excluded
because the R2 values for some enzymes decreased remarkably
there (Fig. 1A, D, and 1G).
At each estimation plots, the absolute deviations of Km estimates
increased in proportion to [E]0 (Fig. 2A), while interestingly, they
were dependent on relationship between k1r and k2 (Fig. 2B).
Especially, (Kma-Km)/[E]0 varied with the ratio of k1r to k2

Table 3
The ranges of log10(Km/[E]0) for estimating Km or Vmax with relative deviation below a% for all the enzymes.

log10 ([S]0 /Km) 0.5e0.5 1e1 3e3

a(%) 5 10 5 10 5 10

Hanes Km 2.5e3.0 2.0e4.5 2.0e3.0 2.0e3.5 2.0e2.5 1.5e4.0

Vmax 1.5e3.5 1.0e4.0 1.0e3.5 0.5e4.0 0.0e3.5 0.0e4.0
Lineweaver-Burk Km 2.5e3.0 2.0e4.0* 2.5 2.0e2.5 e e
Vmax 1.5e3.5 1.0e3.5 2.0e3.5 1.5e3.5 e e
Eadie-Hofstee Km 2.5e3.0 2.0e4.0* 2.5 2.0e3.0 e 2.0e2.5
Vmax 1.5e3.5 1.0e4.0 1.5e3.5 1.0e4.0 0.5e3.5 0.0e4.0

* The Km estimates for some of enzymes with Km & 1.1  104mM and kcat&103min1 deviated by 10e12% at log10(Km/[E]0) ¼ 3.5.
16 K.-I. Yun, T.-S. Han / Biochimie 176 (2020) 12e20
Fig. 2. Absolute deviations of Km estimates within the range of [E]0 ¼ (10¡2~10¡0.5)Km depend on initial enzyme concentrations and the ratios of k1r to k2. In A, B, C, the
range of [S]0 is (100.5e10 0.5)Km. B and C are the results by Hanes plot. And C is the result with respect to log10(Km/[E]0).
regardless of these absolute sizes, and changed rapidly within log10
(k1r/k2)¼(-1e1) but little over the rest range (Fig. 2C). In Fig. 2C,
there were some outliers for some of the enzymes with
Km z 104mM and there were similar results for the other [S]0
ranges and for the other estimation plots (See Table S1~S3).
From Fig. 2 and Table S1~S3, Kma can be described as a function
of [E]0 within [E]0¼(102e100.5)Km:
Kma ¼ Km þ k½E0 ;
In Eq. (6), k is a dimentionless parameter varying with the ratios
k1r/k2 and Km/[E]0, and the range of [S]0.
In Fig. 2C and Table S1~S3, the distribution of the parameter k
narrowed as [E]0 converged to Km. At each [S]0 ranges, distributions
of k values were narrowest by Hanes plot but broadest by
Lineweaver-Burk plot. Nevertheless, the k values mainly depended
on the ratio k1r/k2, and those for k1r [ k2 were smaller than those
for k1r & k2. Interestingly, in case of Eadie-Hofstee plot, k was
nearly 1 at k1r [ k2 regardless of the [S]0 ranges (Table S2). In case
of Hanes plot, k was nearly 1 only at the range [S]0¼(100.5e100.5)
Km but decreased towards about 0.5 with increasing the range of
[S]0 and the ratio k1r/k2 (Table S1).
It is important to note that Eq. (6) was formulated indepen-
dently with Vmaxa.
On the other hand, the parameter k should not be disccused at
[E]0<0.01Km, because the k values there varied very broadly, even
were negative, and further did not carry consistent information on
the ratio k1r/k2.
If necessary, one could draw the constraint jkj, ½E0 = Km  0:1
since most of Km estimates deviated below 10% at [E]0<0.01Km.
3.3. There is a novel comprehensive approximation for enzyme
assays: case study
Assuming Vmaxa z Vmax due to much smaller deviations of Vmaxa
in comparison to those for Kma at a certain range of [S]0 (Fig. 1~2 and
Table 3), Kma and Vmaxa have a linear relationship within
[E]0¼(102e100.5)Km from Eq. (3) and Eq. (6):
Kma ¼ Km þ ðk = kcat Þ,Vmaxa
From the Eqs. (1)~(3), (5), and (6), the dynamics of HMM-system
at [E]0<Km can be approximated as below:
kcat ½E0 ½S0 =ðKm þ ½S0 Þ ; ½E0  0:01Km
v0 ¼ f
kcat ½E0 ½S0 =ðKm þ k½E0 þ ½S0 Þ ; 0:01Km  ½E0 < Km
Eq. (8) intuitively shows that the HMM-system can be
approximated as relationship between [S]0 and v0 at [E]0<Km, so
that one can perform enzyme assays even at the enzyme concen-
trations beyond the upper bound of [E]0 for validity of MM
equation.
We compared fitness of the new approximation for HMM-
system at very broad ranges of initial conditions: [E]0¼(103e10 )
Km and [S]0¼(105e105)Km with those of MM equation and PEA
model, respectively, where the stationary version of PEA model
(6) used for parameter estimation is described as the following [18].
Vmax ½S0 =ðKm þ ½S0 Þ ; ½E0 < ½S0
v0 ¼ f (9)
Vmax ½S0 =ðKm þ ½E0 Þ ; ½E0 > ½S0
As shown in Fig. 3, the estimation deviations for initial velocity
v0 of HMM-system varied with each approximations and with
respect to the ratios Km/[E]0 and k1r/k2.
In each subgraphs, the deviations at log10(Km/[E]0)S2.0 were
nearly identical to each other and trivial, especially below 10% at
conditions [S]0S103Km. Note that both of MM equation and PEA
model generated ideal results at log10 ([S]0 /Km)S1.0: [S]0S10Km at
any initial enzyme concentration.
At the initial conditions log10(Km/[E]0)<2.0 and [S]0¼(103e103)
Km, the deviation degrees had a tendency to increase along with
[E]0, but they varied with the approximation types and the ratios
k1r/k2. At [S]0zKm, real velocities were underestimated by the MM
equation (A1~A3) and the PEA model (B1~B3), but overestimated
by Eq. (8)(C1~C3). At each conditions of log10(Km/[E]0)<2.0, the
results of PEA model were much better than those of MM equation
but inferior to those of Eq. (8), especially for the cases of k1r ¼ k2
and k1r ≪ k2. At all the ratios k1r/k2, the estimation results by Eq.
(8) were best at the conditions of log10(Km/[E]0)>0, still much better
than those by the PEA model even at [E]0 ¼ Km. In addition, Eq. (8)
has room of further improvement by tuning the parameter k.
After all, Eq. (8) is most desirable to approximate the dynamics
of the HMM-system, thus allowing to characterize enzymes at
broader initial conditions: [E]0<Km and [S]0¼(103e103)Km for all
MM-type enzymes.
4. Discussion
(7)
In the paper, we investigated the correlation between HMM-
system and MM equation focusing on relationship between initial
enzyme concentration [E]0 and Michaelis constant Km and obtained
much comprehensive information in connection with enzyme
(8) assays.
In the previous literature [5], the upper-bound of [E]0 for validity
of the MM equation at all the substrate concentrations within very
 K.-I. Yun, T.-S. Han / Biochimie 176 (2020) 12e20 17

Fig. 3. The estimation results for initial velocities of HMM-system at different initial conditions. In each subgraphs, line colors represent log10(Km/[E]0). Approximations are
A1~A3: MM equation, B1~B3: PEA model, C1~C3: Eq. (8). The rate constants are A1, B1, C1: (k1, k1r, k2)¼(107, 106, 104); A2, B2, C2: (k1, k1r, k2)¼(107, 105, 105); A3, B3, C3: (k1, k1r,
k2)¼(107, 104, 106), respectively. The k values in Eq. (8) were set as 1 in A1, B1, C1; 1.5 in A2, B2, C2; 2.0 in A3, B3, C3, respectively.

broad ranges of [S]0:(102e103)Km was depicted as [E]0zKm ac-
cording to the QSSA while as [E]0z0.1Km according to the RSA,
where the latter was preferred for accurate parameter estimation.
However, the results in the paper proves that the upper bound
of [E]0 for validity of MM equation in enzyme assays is much lower
than the above bounds.
As shown in Fig. 1, the MM equation, more exactly Eq. (5) suf-
ficiently fitted the dynamics of HMM-system at [E]0<Km as the R2
values obtained by the three linear transformations of MM equa-
tion were nearly 1 for all the enzymes. Accordingly, the dynamics of
HMM-system can be described with the relationship between [S]0
and v0. But it does not mean that the estimates for Km and Vmax are
always equal to their true values. Indeed, the estimates varied with
the ratios of Km to [E]0, the ranges of [S]0, and the estimation plots,
respectively. As shown in Table 3, the upper bound of [E]0 for
estimating the parameters simultaneously at a certain range of [S]0
for all the enzymes was one percent of Km.
For all that, the parameters in the MM equation cannot be
necessarily estimated only at [E]0&0.01Km.
As shown in Fig. 1, at [E]0S0.01Km, the relative deviations of
Vmax estimates were much smaller than those of Km estimates and
further decreased with increasing the range of [S]0 at [E]0zKm.
Since most of them were around 10% in case of Hanes plot, it would
be reasonable to adopt Vmaxa z Vmax at [E]0<Km. While, the relative
deviations of Km estimates at [E]0S0.01Km increased in proportion
to [E]0 at all the [S]0 ranges and estimation plots (Fig. 2). And the
proportional coefficient k varied not only with the ratio Km/[E]0, the
[S]0 range, and the estimation plots, but especially wth the ratio
k1r/k2 (Table S1~S3). Thus, the relationship Kma ¼ Kmþ0.5 [E]0 at
high enzyme concentrations in the literature [28e31] describes
special cases (Table S1). Significant deviations of Km estimates and
remarkable disparities among different transformations of the MM
equation at [E]0S0.01Km confirms that the upper bound of [E]0 for
validity of MM equation is one percent of Km. Anyway, one can
estimate Km accurately by changing [E]0 within a certain range
according to Eq. (6) or (7) as long as condition 0.01Km&[E]0<Km is
hold.
Coordination of the above discussion is just Eq. (8). This equa-
tion is a most comprehensive approximation for the HMM-system,
because it explains enough about the kinetic behaviors of diverse
enzymes at broader enzyme concentrations.
Given that the parameter k in Eq. (8) be 1 at the condition
k1r [ k2 (Fig. 2C, Table S1~S3), Eq. (8) becomes similar to the
stationary version of PEA model: Eq. (9), which is originated from
the overall PEA model at the condition Ks/Km ¼ 1 [18]. That is why
Eq. (9) can be used only at k1r [ k2. Additionally, subequations in
Eq. (9) have different scopes only based on the relationship be-
tween [E]0 and [S]0. In fact, the adoption criterion is not strict ac-
cording to the results of A1~A3 in Fig. 3, where the MM equation
approximated the dynamics of HMM-system at the initial condi-
tions of [E]0&0.01Km, [S]0S103Km and futher generated the
tolerable results within the range of [S]0¼(105e103)Km as well.
The results of Eq. (8)(Fig. 3C1) were much better than those of Eq.
(9)(Fig. 3B1) even at k1r [ k2. Eq. (8) safisfactorily approximated
18 K.-I. Yun, T.-S. Han / Biochimie 176 (2020) 12e20
the dynamics of HMM-system at the initial conditions of [E]0<Km,
[S]0¼(103e103)Km for all the relationships of k1r and k2(C1~C3 in
Fig. 3).
There is also the MM-like approximation based on tQSSA [10,11],
or ECA model [15e17]. The equations are expressed identically as
the following.
d½ST = dtzVmax ½ST =ðKm þ ½ET þ ½ST Þ; (10)
where [E]T is total enzyme concentration: [E]T ¼ [E]0, and [S]T is
total substrate concentration ([S]T ¼ [S]þ[ES]).
Eq. (10), as an improved approximation for the HMM-system, is
valid not only at the more stringent condition [E]0≪[S]0þKm for
validity of MM equation, but also at condition [S]0≪[E]0þKm for
validity of RMM equation [13,14], but it is not suitable for enzyme
assays due to the presence of [S]T that is usually impossible to
measure. Again, it is also satisfied at k1r [ k2 [40].
Conventionally, the plausible hypothesis of k1zk1r [ k2
seems to be prevalent in enzyme kinetics [25]. Though, there are
obvious alternatives: k1r z k2 for typical isomerases [34] and
k1r ≪ k2 for some “superefficient” enzymes [41,42] or according to
the reaction direction. Under the circumstances, the universal Eq.
(8) is prefer to Eq. (10).
In the paper, we elucidated the possibility of drawing more in-
formation on enzyme characteristics, apart from the traditional
parameters Km and Vmax, through initial rate experiments.
The estimation deviations for Km at 0.01Km&[E]0<Km depended
on the relationship between k1r and k2 and [E]0 (Fig. 2), indicating
that differences in kinetic behaviors according to the ratio k1r/k2
are distinguished in the curve of [S]0~v0 only at 0.01Km&[E]0<Km.
The novel parameter k mainly varied with the ratio k1r/k2 was
also different along with the ratios of Km to [E]0, the [S]0 ranges, and
estimation plots (Fig. 2, Table S1~S3). So, one can estimate the k
value within a certain range of [S]0 using a suitable estimation plot
to determine the relationship between k1r and k2 and further to
confirm whether the estimated Km is a measure of substrate affinity
(Ks ¼ k1r/k1), or not. The range of 0.03Km&[E]0&0.3Km appears
suitable for estimating the parameter k, for the distributions of k
values get broader with decreasing [E]0 as shown in Table S1~S3.
Let’s call it as “the k zone” hereafter.
According to Eqs. (2), (7) and (8), the three factors: [E]0, k, and kcat,
condition each other in estimating them, and thus one should know
at least one of them to complete the assay. The fundamental problem
is that the enzyme characteristics (rate constants k1, k1r, and k2, or
even [E]0) might remarkably change according to enzyme amount
for several reasons in vitro or in vivo. No one can assure that the
parameters Km and Vmax estimated at [E]0&0.01Km are equal to those
at [E]0zKm, so it would be necessary to perform enzyme assays
taking into consideration the real environment of the enzyme in an
artificial or living cell. Still, the value of log10 (k1r/k2) could change
little if the reaction conditions besides enzyme amount were
balanced. In this sense, the parameter k is a characteristic constant
for enzyme assays. Very narrow distribution of k values allow to
estimate both of [E]0 and kcat within more specific ranges by Eq. (6) or
(7). Further, knowledge of the rate constants k1r and k2 for the same
or similar enzymes would result in the perfect estimates.
Eventually, the k zone would be an arena or a “golden” zone in
the field of enzyme kinetics.
Eq. (8) can offer more reliable assay than the (EA)2 assay [18].
It is possible to estimate Ks(¼k1r/k1) by the (EA)2 assay but it
needs a kinetic experiment at [E]0 ¼ Km. The reason can be
explained separately and sufficiently by Eq. (8); The experiment
curves only at [E]0S0.01Km reflect the relationship between k1r
and k2. But the PEA model, Eq. (9) generated the results poorer than
those of Eq. (8) even at k1r S k2 and especially the estimation
results at [E]0S0.1Km were not desirable (Fig. 3B1 and 3C1). In
addition, Eq. (8) generated much better results at [E]0 ¼ Km. Eq. (8)
is also useful at k1r z k2 or k1r ≪ k2 (Fig. 3C2 and 3C3).
And the (EA)2 assay may be exposed to product inhibition and is
hardly applied to more complicated enzyme reactions, because it
needs measuring characteristic time constant t∞ during late reac-
tion phases, whereas Eq. (8) is versatile like the MM equation in
enzyme kinetics just as based on initial rate experiment.
All the above discussion proves that Eq. (8), as a comprehensive
approximation for the HMM-system, is most powerful in enzyme
assays.
Intuitively Eq. (8) concretizes some indefinite concepts in
enzyme kinetics.
As shown in Eq. (8), the dynamics of HMM-system at [E]0<Km
can be approximated differently within the two intervals of enzyme
concentrations. Enzyme kinetics at “low enzyme concentrations”:
[E]0&0.01Km is the MM equation. But enzyme kinetics at “high
enzyme concentrations”: 0.01Km&[E]0<Km obeys a new equation.
Thus, Eq. (8) makes a substantial concept of the uncertain one
for “high or low enzyme concentrations” in previous literature
[11,18,20,28e32] by focusing on the characteristic constant Km. The
results of Fig. 3 illustrate well that enzyme kinetics vary with the
ratio [E]0 /Km rather than the ratio [E]0/[S]0. In fact, Eq. (9) was
implicitly established at 0.01Km&[E]0&Km [18].
vOf course, there is a constraint between [E]0 and [S]0 in enzyme
assays. The fact that the MM equation cannot approximate the HMM-
system at [E]0[[S]0 even when RSA is valid was proved theoretically
[43]. Actually in Fig. 3, too, the deviations at [S]0&104Km were sig-
nificant, but they varied with the approximations, and the ratios Km/
[E]0 and k1r/k2. Nevertheless, the HMM-system agreed with Eq. (8) at
[S]0S103Km at least in Fig. 3. The substrate concentration of
[S]0 ¼ 103Km seems enough low in discussing the enzyme kinetics.
From the viewpoint, quantitative relationship between [E]0 and [S]0 is
no longer essential in enzyme assays.
Also, the physical meaning of Michaelis constant Km should be
reestablished according to Eq. (8).
Generally, Km is said to be the substrate concentration at
v0 ¼ Vmax/2, but the statement is valid only at low enzyme con-
centrations: [E]0&0.01Km, as shown in Eq. (8). Obviously, Km gov-
erns the limits of each enzyme kinetics in Eq. (8), and such a
statement would reflect a real meaning of the characteristic con-
stant. In the other words, Km is the upper bound of [E]0 for enzyme
assays by using Eq. (8), and such a definition is more specific and
practical than the meaning of Km in relation to the steady-state [44].
In the future, it is necessary to investigate the possibility of char-
acterizing an enzyme at enzyme concentrations beyond Km for sure.
Although there are many methods for estimating the parame-
ters of MM equation [25], we used the typical linear trans-
formations in the paper.
The merits or shortcomings of the transformations have been
already discussed [45], and also reproduced in all the results in the
paper. Again, Hanes plot was best and Lineweaver-Burk plot was
worst, while the R2 values by Eadie-Hofstee plots remarkably
reduced at inapplicable conditions. The paper results proves that
Hanes plot is still good even at high enzyme concentrations. And as
shown in Figs. 1 and 2, all the three transformations deviated at
[E]0S0.01Km, proving that the assumption of [S]z[S]0 is not valid
there.
In Tables 1 and 2, there are some enzymes with the estimation
deviations beyond 10% at [E]0&104Km, and the kind of enzymes
varies with the [S]0 ranges and estimation plots. The necessity of
the estimation deviations at [E]0&104Km must be futher
researched but could be related with the computational errors to
some extent. But, in case of Hanes plot and [S]0¼(100.5e100.5)Km,
the estimates only for some enzymes with Km below a few mM
 K.-I. Yun, T.-S. Han / Biochimie 176 (2020) 12e20
deviated significantly at [E]0<104.5Km (Table 3). Therefore, the
lower limit of enzyme assays would be less significant even in the
theoretical aspect.
Anyway, Hanes plot and [S]0 ¼ 100.5e10 .5Km generated best
results in Table 3, which suggest a robust procedure for usual
enzyme assays, while it will be necessary to measure initial ve-
locities at [S]0z10Km for accurately estimating Vmax within the k
zone (Table 3, Fig. 3).
All the results and discussion in the paper are originated from an
assumption that enzyme characteristics could change little at any
enzyme and substrate concentrations. In real enzyme assays, there
are many limitations including accurate measurement of initial
velocities at high enzyme concentrations and different kinds of
interferences or errors. Furthermore, the validity of Eq. (8) must be
proved analytically and more firmly.
However, the novel approximation, Eq. (8), would be a stepping-
stone in the development of theoretical and practical enzyme
kinetics.
5. Conclusions
At the initial enzyme concentration [E]0 below Michaelis con-
stant Km, the dynamics of the HMM-system can be approximated as
the relationship between initial substrate concentration [S]0 and
initial reaction velocity v0. The MM equation is valid at low enzyme
concentrations: [E]0&0.01Km, while enzyme kinetics at high
enzyme concentrations follow a new equation, thus allowing to
characterize enzymes more comprehensively.
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Acknowledgements
We sincerely appreciate the advices of the anonymous re-
viewers and the editor.
19
This work was supported by KIM IL SUNG University(Project No.
19-85-02).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.biochi.2020.06.002.
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