196
PII: S 0 3 0 7 - 4 4 1 2 ( 9 6 ) 0 0 0 8 9 - 1 programs in FORTRAN which included routines for cal-
Current Statistical M e t h o d s for Estimating the Km
and Vma~ of M i c h a e l i s - M e n t e n Kinetics
RAYMOND J. RITCHIE 1 and TANIA PRVAN 2
~Botany Department
School of Biological Sciences
The University of Sydney
Sydney, NSW 2006
Australia
and
2Statistics and Operations Research Unit
School of Mathematical Sciences
University of Technology, Sydney (UTS)
Broadway, NSW 2007
Australia
Introduction
Methods for determining the Km and Vma~ of the
Michaelis-Menten equation (eqn 1) and similar rect-
angular hyperbolae, are very important to modern
biology, l-s° Much effort has been put into developing
mathematically rigorous methods for obtaining the 'best'
estimates of I ~ and Wmax.4'6'7'10-14 Most standard bio-
chemistry texts do not warn students that estimates of K~
and Vmax have inherently large errors, even though the
statistical limitations of the Km and Vma~parameters have
been known for a long time. ~'2'4'5'15
Calculation of the variances or standard errors of Km
and Vm~, has generally been neglected, even though
methods for determining them have long been avail-
able. 1'2'4"s'8'1°'14-16It is just as important to know the errors
of estimates of Km and Vm~x as it is to have the 'best'
estimates of these parameters. The statistics of I ~ and
Vmax are critical to the identification of competitive and
non-competitive inhibition because these phenomena are
defined in terms of significant effects upon Km and/or Vma~
of an inhibitor. 6 Without valid estimates of Km and Vm~
and their errors, it is not possible confidently to identify
inhibitors or what class of inhibitor they belong to.
Calculation of standard errors of parameters of non-
linear equations is a routine part of modern statistical
packages such as Minitab ® and SigmaPlot®. Dowd and
Riggs 4 warned that computer simulations showed that the
errors of Km and Vm~x,determined using double reciprocal
and Hofstee plots were often very large and heavily
biased. Cleland ~ discussed the use of least squares fitting
methods to estimate Km and Vma~and matrix algebra to
calculate their errors, but at the time such techniques
were unsuited to routine work or class work. Cleland 2
later published a wide selection of non-linear least squares
Abbreviations: E(V~), is the expected velocity at substrate concentration
(i), K~, the Michaelis constant, MSRes, mean square residual, r, correla-
tion coefficient, s2, variance, SE, standard error of the mean, [S~], the (i)th
substrate concentration, SSReg, sum of squares of the regression, SSRes,
sum of squares residual, SST, total sum of squares, V~, the (i)th velocity
measurement, Vm=, the saturating velocity
BIOCHEMICAL EDUCATION 24(4) 1996
culating the errors of the fitted parameters. Detailed dis-
cussions of least squares fitting techniques applied to
biochemical problems are available. 2'5'9'15-I7
An important aspect of the problem of making reason-
able estimates of Km and Vmaxis to appreciate the limita-
tions imposed by routine biochemical practice. Our recent
simulation studyis showed that for realistic levels of
experimental errors in measured velocities, at least 10
data points were needed for estimates of I ~ to be nor-
mally distributed. A survey of current practice showed
that the mean number of data points being used in pub-
lished data is about sixTMwhich is no improvement over
practice 30 years ago? Typical biochemical kinetic data
sets are therefore small. This makes more sophisticated
statistical methods such as 'bootstrap '19 and various 'jack-
knife' methods 2° either inapplicable or not providing any
theoretical advantage over the non linear least squares
method.
Parametric statistics are the more familiar statistics
used by biologists. Non-parametric fitting methods have
been developed. "'21 23It is possible to calculate confidence
limits for median estimates of Km and Vmax.24-26 Since for
normally distributed data, the median and mean are not
significantly different, non-parametric methods are a con-
venient check for estimates of parameters made using
parametric methods and will give usable estimates of I ~
and Vmaxunder the widest experimental conditions. Non-
parametric estimators need to be treated with caution: for
example, it is not valid to calculate an overall mean and
error of a set of median estimates of I ~ . A non-para-
metric estimate of I ~ must not be confused with a para-
metric estimate.
The ready availability of statistical packages is not
entirely a good thing because statistical packages are now
often used with no visual inspection of the data and the fit
obtained is often taken at face value 5. The user might not
be made aware of the statistical limitations or validity of
weighting routines incorporated into commercially avail-
able p a c k a g e s . 2'5'1°'27
Theory
The Michaelis-Menten equation is,
[S]Vm~
v (1)
where, V is the velocity of the reaction, Vm~ is the satura-
tion velocity, [S] is the concentration of the substrate and
I ~ is the Michaelis constant. Eqn (1) has an error term
(el) for the measured velocities. In this paper we have
assumed that it is a statistically independent, normally
distributed error.
Difficulties in making accurate determinations of I ~
and Vm~, from simple graphs of velocity (V) vs [S] led to
the extensive use of three linear transformations of the
Michaelis-Menten equation, the double reciprocal plot

(eqn 2), the Hanes-Woolf plot (eqn 3) 28'29and the Eadie-
Hofstee plot (eqn 4). 29
1 Km 1 but is really no more than an a d h o c approximation.
- - - +-- (2)
V Vm~[S ] Vm~
IS] 1 Km
-- IS] + - - (3)
V Vmax V ....
KmV
V= --- +Vm,x (4)
[s]
The double reciprocal, Hanes-Woolf and Eadie-
Hofstee plots allow the determination of Km and Vm~x
from straight lines of best fit, usually fitted by linear
regression using unweighted data. However, all these
transformations impose limitations not found when using
a non-linear fit to eqn (1). The double reciprocal and
Hanes-Woolf plots (eqns 2 and 3) both involve the
inverse of V and eqn (4) the inverse of [S]. Zero or even
negative velocity values are valid data for eqn (1) but 1 / V
and [S]/V are both undefined if V= 0 and V/[S] is unde-
fined for IS] = 0. If the calculation of velocities involves
the subtraction of a control zero (for example a spon-
taneous breakdown rate of a substrate), occasional small
negative velocities will be found in data sets. Such values
are valid data if a non-linear least squares fit is used but
cannot be used if eqns (2) or (3) are used for fitting.
Selwyn3° discusses non-linear curve fitting to a Michaelis-
Menten curve where there is a non-zero velocity at zero
substrate concentration.
The error terms in eqns (2), (3) and (4) are all different
from (el) and so give biased fits, when compared to a non-
linear least squares fit (eqn 1). The double reciprocal plot
is still the most popular linear transformation even though
it is known to be the most unsatisfactory method for deter-
mining K~. 1'2'4'5'13'14 The y intercept of the graph is 1/Vm~x
and so the standard error of l/Vine, (SE(1/Vm~)) can be
calculated by using the standard formulae for calculating
the error of the y-intercept of a linear regression.25'26 Vm~,
and the standard error of Vmax (SE(Vm~x)) are calculated
by inversion of 1/Vmaxand its error; the resulting standard
error of the Km is only approximate. The Km constant can
be read off a double reciprocal plot as the x-intercept
(-1/Km). However, the calculation of the error of the
x-intercept of a linear regression results in error-bars
which are asymmetrical25'26 making the estimation of the
errors of the inverse of 1/Kin impractical. The most prac-
tical way of calculating the Km and SE(Km) is to divide the
slope m of the double reciprocal plot (Km/Vm~x)by 1/Vm,x.
Estimates of SE(Km) will contain errors contributed by the
errors of both K J V .... and X/Vmax.
The bias of the unweighted double reciprocal plot can
be partially corrected by assigning the correct weighting to
the elements of the set of data. There are several opinions
on the most appropriate weighting.2'31'32 The most com-
monly used weighting of each data pair (1/[Si], l/G) is the
BIOCHEMICAL EDUCATION 24(4) 1996
197
fourth power of the velocity (V?), where (i) is the (i)th
data value. 3'4'1° ~2.2v,32This weighting dates back to Line-
weaver, Burk and Deming 34 and Lineweaver and Burk 33
The Hanes-Woolf plot is rarely u s e d . 28'29'35 It is a semi-
reciprocal plot of [S]/V vs [S]. The independent variable
appears on both sides of the equation but the x-axis is IS]
not 1/[S] as in the double reciprocal plot. Vm,xand Km are
not read directly off the plot, the y-intercept is Km/gma x
and the slope m is l/Vma, the errors of which can be
calculated by standard linear regression methods. 25'26Only
approximate calculations of the errors of Km and Vma~can
be made (as for the double reciprocal plot). The primary
advantage of the Hanes-Woolf plot is that a plot of [S]/V
vs IS] is not as heavily biased by velocities at very low
substrate concentrations as the double reciprocal plot.
The Eadie-Hofstee Plot 29is a semi-reciprocal plot of V
vs V/[S] (eqn 4). The advantage of this linearization
method is that Vm~xand Km can be read directly off an
Eadie-Hofstee plot, the y-intercept is the Vm~ and the
slope m is -Km. The fundamental disadvantage of the
Eadie-Hofstee equation is that Vappears on both sides of
eqn (3); V/[S] is not an independent variable. This is a
serious and unfortunately, intractable, statistical problem.
A standard linear regression of Y upon X assumes that X
is an independent variable, measurable without error, and
Y is a function of X, with a normally distributed e r r o r . 25'26
The use of a least-squares linear regression technique to
determine Km and Vmaxfrom a plot of V vs V/[S] violates
the inherent assumptions used to calculate the least-
squares linear regression fit.
More recently, Studnicka7 published a method he
termed 'hyperbolic regression'. The method does not treat
IS] as the independent variable and V as the dependent
variable with a constant error variance. The fitting method
used by Studnickav is not a valid least squares fit because
the covariance associated with the fit is a function of [S]
and the unknown K~. Since the variance is a function of
IS], the least squares approach is inappropriate. Fortu-
nately, the method has not come into widespread use, but
could find its way into a biochemical statistics package.
Non-linear curve fitting to the Michaelis-Menten
equation
The general availability of personal computers means that
tedious iterative procedures involved in non-linear least
squares fitting of curves are no longer a deterrent to their
routine use. Least-squares fitting routines, using calculus
and matrix inversion, incidentally provide estimates of the
errors of Km and Vmax.Asymptotic standard errors of Km
and Vm~,were calculated as previously described, 1'2'5'~5the
limitations of which are discussed by Johnson and Faunt 5
and Straume and Johnson? 7 Standard routines are avail-
able in common computer languages used on personal
computers (FORTRAN, BASIC, PASCAL and C). Some
commercially available statistical and graphics packages,
for example SigmaPlot®, use the more rapidly converging
198
and more 'rugged' Marquardt algorithm instead of the
Gaussian Least Squares m e t h o d . 5'36
Programming and methods
The BASIC program was developed by the authors using
Microsoft® QuickBASIC for Apple ® Macintosh ® Systems
Version 1.0 (Binary Math) (Microsoft Corporation, Red-
mond, Washington State 98073-9902, USA) run on a
Apple ® Macintosh ® computer with 4.0 Meg RAM. The
non-linear least squares fitting routine is available in com-
piled or code forms in MicroSoft BASIC for Macintosh.
The MonteCarlo simulator is also available for teaching
purposes. Our own BASIC implementation of the non-
parametric Cornish-Bowden fitting method H is also avail-
able upon request.
Most commercially available statistics packages are
capable of non-linear least squares fitting but may not
necessarily calculate the errors of the fitted parameters.
We have found the following packages to be fully capable
of non-linear least squares fitting and do calculate the
errors of the fitted parameters; Minitab 8®by Minitab Inc.
(Pennsylvania State College, PA 16801-3008, USA), Sig-
maPlot ® by Jandel Scientific Corp. (San Rafael,
California 94901, USA).
Non-linear least-squares fit to Michaelis-Menten equation
The program was made up in two parts. The first part is a
generalized least squares fitting program. 3 Similar
updated versions of this program are available in FOR-
TRAN, PASCAL and C31'32and in B A S I C . 37 The second
part is a matrix inversion fitting routine that refines the
estimates of I ~ and Vm~xand allows estimations of the
errors of these estimations of Km and Vmax. Cleland 2 also
used a two-step procedure for his FORTRAN routines.
The random search method of Tseng and Hsu TMshould
give equivalent results to the above, as it too is a search
routine followed by matrix inversion.
The least-squares procedure used was very robust and
searches for new estimates of Km and Vm~xin a stepwise
fashion while the sum of squares residual, or SSRes, is
reduced. 3 The magnitude of the SSRes is not used, only its
relative size. The program calculates the square of the
difference between the observed and calculated V values
for given values of Km and Vmaxfor all the given values of
[S~] and then calculates SSRes,
Vmax[Si] ~2
SSRes = i=1
~' Vi ~---~N/] (5)
where, N is the number of data points, V~is the observed
velocity at [Si] and an expected velocity, E(V~), is cal-
culated using the current estimates of Km and Vm~x.The
program searches both upwards and downwards for more
favourable values of Km and Vmaxby adding or subtracting
an increment to Km and Vm~xand calculating the resulting
SSRes. The solution for Km and Vmaxis not necessarily the
optimum fit, it is the first SSRes minima or 'pit' encoun-
tered in the Km/Wmax space? This is usually not a great
BIOCHEMICAL EDUCATION 24(4) 1996
problem if only two parameters are to be fitted, but
becomes a major problem when there are three or more
fitted parameters, for example a Michaelis-Menten curve
with a constant non-enzymatic background rate for a
reaction. 3°
The Km and Vmax values obtained using the 'Pattern
search' sub-program were then used as the starting point
for a second iterative least squares procedure using cal-
culus and matrix algebra (Gauss-Jordan Matrix Inver-
sion) to obtain progressively refined values for Km and
Vm~xand estimates of their variances. 2'5 Once the optimum
values for Km and Vmax (minimum SSRes, eqn 5) have
been found, the final SSRes is calculated as well as the
sum of all Si and V~values and the sum of V~2. Since two
parameters are calculated from the data, the Mean
Squares Residual (MSRes) has N - 2 degrees of freedom
(df). The correlation coefficient (r) is calculated as the
square root of the ratio of SSReg to SST and is defined
from r 2= 0 (no correlation) to r 2= 1 (perfect fit).
The diagonal elements of the inverse matrix, M-l(1,1)
and M-1(2,2), are then used to calculate the standard
errors of the mean estimates of Km and Vmax,2'5'8 Sigma-
Plot ® and Minitab ® use the same method for calculating
the standard errors of fitted parameters.
SE(Km) = X/M 1(1,1)'MSRES (6)
SE(Vmax) : x / M I(2,2)'MSRES (7)
Eqns (6) and (7) have been included because some
statistical packages give the solution matrix for a non-
linear curve fit [M 1(1,1) to M-l(n,n)] but the user is
expected to know how to calculate the standard errors of
the fitted parameters from this covariance matrix.
Finally a mean, variance SSRes, Total SS (SST), mean
square residual (MSRes) and a correlation coefficient (r)
is calculated.
Results
Example data No 1
Table 1 gives some sample data, originally from Atkinson,
Jackson and Morton 4°. These data have been used before
as sample data in discussions of statistical estimations of
the errors of Km and Vmax6'10. We have used three signifi-
cant figures in all the Tables. The set of substrate concen-
trations was spread out in an approximately inversely
Table 1 Sample Data No 1 for Comparison of Different Methods
of Estimating Km and Vm~.A set of sample enzymatic kinetic data,
originallyfrom Atkinson, Jackson and Morton 4°for comparison of
various methods for estimating K,~ and V,~ The approximate
position of the Km is shown by an arrow
Substrate [S] Velocity V
0.138 0.148
0.220 0.171
0.291 0.234
0.560 0.324
0.766 0.390
1.460 0.493

Table 2 Unweighted and Weighted Double Reciprocal and Eadie-Hofstee Plots Compared to a Non-Linear Least Squares Michaelis-
Menten Fit. Comparison of estimates of Kin and Vm~ using different fitting techniques using the data set shown in Table 1
Lineweaver-Burk Fit Hanes-Woolf Fit
K,, _+SE 0.441 _+0.0906 0.583 _+0.0523
V.~ _+SE 0.585 _+0.104 0.685 _+0.0384
Regression (r) 0.979 0.994
Probability (p) 0.06% 0.006%
SSRes* 2.88 X 10 -3 747 x 10 6
*SSRes calculated for fit of untransformed data to Michaelis-Menten equation.
proportional manner, with about the same number of data
points above and below the Kin. The correlation coeffi-
cients (r) and variances (s 2) obtained using transformed,
untransformed data, weighted and unweighted regres-
sions are not easily comparable. For meaningful compari-
sons, the sum of squares residual (SSRes; eqn 5) has been
calculated using the untransformed V~ and [Si] data
(Table 1) and the Michaelis-Menten equation using the
estimates of K~ and Vm~, made using the different
methods. The SSRes provides an objective measure of
how well the variously obtained estimates of Km and Vm~,
fit the untransformed experimental data (the closer to
zero the better the fit).
Double reciprocal plot
By standard linear regression of 1/V upon 1/[S],
- -1 = 0.798 ([-~]) + 1.708
V Vm= and Km calculated using eqns (8) and (9) above
r=0.979, p =0.07%, n =6, 1/Wm~x=1.71-+0.303 (__
standard error of mean)Km/Vmax=0.758±0.0782 (q-
standard error of mean) An approximate error for an
inverse (Z = l/X) is
If covariance terms are ignored, the approximate error
for a division (Z =X/Y) is,
SE(Z)
Using eqns (8) and (9) above, the estimates of Km and
Vm~xare,Kin = 0.441 _+0.0906 ( + standard error of mean)
Vm,x =0,585 _____0.104( + standard error of mean).
The above estimates of Km and Vm~ are very imprecise,
even though the correlation coefficient (r=0.979) indi-
cates a very good fit. If we replace the standard errors by
+ 9 5 % confidence limits by multiplying them by the
appropriate student's t value (t(2,0.05,4) : 2.776), the relative
errors of K m and Vmaxare + 57% and -+ 49% respectively.
Such poor estimates of Km and Vmax are of limited
utility.
BIOCHEMICAL EDUCATION 24(4) 1996
199
Eadie-Hofstee Fit Least Squares Weighted Inverse Transforms
Michaelis-Menten Fit Wilkinson's V i i 4 Weighting
(NB This weighting is not valid)
0.490 _+0.0834 0.597 _+0.0683 0.571 _+0.0481
0.626 _+0.0600 0.690 _+0.0368 0.680 + 0.0352
-0.947 0.996 0.981
0.42% 0.003% 0.03%
1.39 x 10- 3 736 x 10-6 770 x 10 6
The weighted regressions on the double reciprocal
transformed data give estimates of Km and V ..... closer to
those obtained using the non-linear least squares method
(Table 2). If a fit to a data set is unbiased, squared devia-
tions from the fitted line (the residual squares) should be
independent of the dependent variable 17. Pearson's r test
and the non-parametric Spearman's R H O test 25'26 both
showed that weighted deviations from the fitted line for
the data was not correlated with the velocity (V~).
Weighted deviations from the fitted line using VJ
weighted data 1°'32 was significantly correlated with Vi
( R H O test p < 2 % ) and so the resulting Km and Vmax
values were biased, even though the SSRes was lowest of
the weighted linear fits (0.7 x 10 3).
Hanes- Woolf plot
1 / g m a x and K m / g m a x w e r e determined by standard linear
regression of [S]/V upon IS], then approximate errors for
(Table 2). These estimates of Km and Vm~xare much more
precise than those obtained using the double reciprocal fit
and very close to those obtained using the non-linear least
squares fit. If we replace the standard errors by +_95%
confidence limits by multiplying them by the appropriate
students t value (t~2,00s,4~= 2.776), the relative errors of K m
and V~ax are +_25% and ___16% respectively. This is a
(8) great improvement over the double reciprocal method.
These errors are slightly smaller than those using the non-
linear-least squares method, however the SSRes is larger
showing that the H a n e s - W o o l f method is inferior to the
non-linear least squares method.
Eadie-Hofstee or Hofstee plot
The determination of Km and Vma~ using an E a d i e -
Hofstee plot is more straightforward, although obtaining
a line of best fit by a linear regression is not really valid.
The determinations of Km and Vm~ from an unweighted
regression of V upon V/IS] are shown in Table 2. The
Hofstee plot estimates of Km and Vmax have smaller
standard errors than those based on the unweighted
double reciprocal plot but the estimates of Km and Vm~x
are not as close to those obtained using the non-linear
least squares fit as the H a n e s - W o o l f method (Table 2).
Approximate transformations of the y-intercept and the
slope m are not required to obtain the errors of Km and
Vmax. Although the correlation coefficient (r) is not as
200

close to a perfect fit as in the double reciprocal plot, the Vmax= 0.659 (--0.126, + 0.089). These results are closely
apparent errors of Km and Vmax are smaller. comparable to those made using parametric methods.

Non-linear Michaelis-Menten least squares fit

The unweighted least squares fit to the data is shown in
Table 2. The correlation coefficient is much closer to 1
(r=0.9959) indicating a much improved fit. If the
standard errors are replaced by + 95% confidence limits,
the relative errors of K m and Vma~are + 3 2 % and _+15%
respectively. Estimates of K~ and Vm~ and their standard
errors using the commercially available Minitab ® and Sig-
maPlot ® packages were the same as the above.
Analysis of residuals 17 using Pearson's r test and the
non-parametric Spearman's R H O tests 25'26 both showed
that deviations from predicted velocities in the non-linear
Michaelis-Menten fit were not significantly correlated
with the velocity (Vi) (p ,,~40%).
Table 2 compares the estimates of Km and Vmax
obtained using unweighted and weighted double recip-
rocal plots, the H a n e s - W o o l f Plot and the non-linear
least squares methods. The errors for the Hofstee Plot can
only be calculated assuming the term V/IS] has no signifi-
cant error. Comparisons of the various estimates of K~
and Vm,x in Table 2 show that the double reciprocal plot
gave the worst fit and the largest error-bars. On the other
hand, the correctly weighted double reciprocal plot, the
H a n e s - W o o l f and Hofstee plots and the non-linear least
squares fits to the Michaelis-Menten equation yield esti-
mates of Km and V~,~xthat are not significantly different
but vary considerably in precision.
Non-parametric estimates of Km and Vm,x were made
using methods described by C o r n i s h - B o w d e n u'12,23,z4 and
approximate 95% error limits calculated 25"26.Median esti-
mates of Km and Vma~were: K~ = 0.555 ( - 0.156, + 0.215);
Graphs of data and fitted parameters
Fig la and lb present Data Set No 1 plotted as Vvs IS] and
as a double reciprocal plot of 1/Vvs 1/[S]. The best fit (the
non-linear least squares fit) is shown as a solid line and the
worst fit (the double reciprocal linear regression) is shown
as a dotted line on both graphs. It is apparent from Fig 1
that the double reciprocal regression method gives a
poorer fit at high substrate concentrations than the non-
linear fit. This is a result of the reciprocal transformation
(Fig lb).
Example data No 2
Michaelis-Menten type kinetics are often encountered in
studies of ion uptake by plants? ~ Table 3 shows data for
36C1- uptake flux (nmol m 2 s-l) vs [C1-]o (mmol m -3) by
the blue-green alga, Synechococcus R-2 42 to serve as a
second worked example. The set of substrate concentra-
tions were spaced in a logarithmic manner, with most of
the data points below the K~. A Hofstee plot was used to
estimate Km and Vm~ in the original publication? 3 Table 4
shows a comparison of fitting methods using the data in
Table 3. The Hanes-Woolf and Eadie-Hofstee plots both
appear to give very good estimates of K m and Vmax. The
double reciprocal plot gives the worst estimates of Km and
Vmax- The H a n e s - W o o l f technique again seems to give
overly optimistic estimates of the size of the errors of Km
and Vma~- The non-linear least squares fit gives the most
precise estimates of Km and Vm~x.The SSRes of the double
reciprocal fit was 0.511 but weighting the data reduced the
SSRes to about 0.38.

0.5 -(a) (b)

10

0.4
9
• Velocity Data Points
Michaelis-Menten Least
Squares Fit Z "

:/
8 Double R e c i p r o c a l i l t ~ , ,"

7
////i
0.3
6 • jJ
0.2
4
• Velocity Data Points
i,1¢
•8 ~ Michaelis-Menten Least Squares Fit
3
- - Double Reciprocal Fit
0.1 2

jJJ
I i i I i i i I ,( I I I I I I I I i I
0.2 0.4 0.6 0.8 1.0 1.2 1.4 -3 -2 -1 1 2 3 4 5 6 7 8 9 10
Substrate 1/Substrate

Figure la presents Data Set No 1 plotted as Vvs IS]. The best fit (the non-linear least squares fiO is shown as a solid line and the
worst fit (the double reciprocal linear regression) is shown as a dotted line. The double reciprocal linear regression gives under-
estimations of V at higher substrate concentrations. Figure 1b presents Data Set No 1 plotted as 1/V vs 1/[S]. The double reciprocal
linear regression appears to be biased by velocities measured at low substrate concentrations

BIOCHEMICAL EDUCATION 24(4) 1996

 201

Analysis of residuals 17 using Pearson's r test and the N o n - p a r a m e t r i c estimates of the median Km and Vmax
n o n - p a r a m e t r i c S p e a r m a n ' s R H O tests 25'26 b o t h showed (Km=44.6 ( - 4 . 4 0 , +5.54); Vm~x=18.7 (--0.712, +1.44))
that deviations f r o m the fitted line using all the fitting were very closely similar to the m e a n s obtained by using
m e t h o d s were not significantly correlated with the velocity p a r a m e t r i c methods but are little help in identifying the
(<). most satisfactory p a r a m e t r i c fitting method.
Figs 2a and 2b present D a t a Set No 2 plotted as Vvs [S]
Table 3 Sample Data No 2for Comparison of Different Methods and as a double reciprocal plot of 1/Vvs 1/[S]. In this case
of Estimating Km and Vm~. A set of sample data, originally from the fit to the M i c h a e l i s - M e n t e n relationship is so good
Ritchie. 42 This data set is from a study of nutrient uptake by algal that the best (non-linear least squares) and the worst fits
cells and is a case of where the Michaelis-Menten model has been (double reciprocal linear regression) are almost
used to model uptake of a nutrient by living whole cells. The
approximate position of the Km is shown by an arrow. indistinguishable.

Substrate [S] Velocity V Example data No 3

3.568 1.42 T h e third example is a set of data f r o m Tayyab and
7.213 2.51 Q a m a r 35 on the action of papain on milk casein. The data
10.87 3.51
14.44 4.78 set was kindly provided by D r Tayyab (Table 5). T h e
18.18 5.40 substrate concentrations ( m m o l m 3 casein) were
21.93 6.14 approximately evenly spaced and the velocity calculated
29.24 7.72
36.65 8.65 as m m o l m 3 min-1). Most of the data points are above
~K m the Km. T h e data set is an excellent example of the need to
55.33 9.96 plot up data and inspect it visually. 35 C o m p a r i s o n of Figs
109.8 13.28
3a and 3b shows a m a j o r unfortunate consequence of

Table 4 Unweighted and Weighted Double Reciprocal and Eadie-Hofstee Plots Compared to a Non-Linear Least Squares Michaelis-
Menten Fit. Comparison of estimates of K• and Vm~xusing different fitting techniques using the data set shown in Table 3
Lineweaver-Burk Hanes-Woolf Eadie-Hofstee Least Squares Weighted Inverse Transforms
Fit Fit Fit Michaelis-Menten Fit Wilkinson's Vi4 Weighting
(NB This weighting is not valid)
K~±SE 41.8+3.22 44.0 + 1.53 43.6 + 2.61 43.0 + 2.34 42.7 + 1.73
Vm~__SE 17.8 4-1.34 18.5 +0.461 18.5 +0.753 18.4_+0.506 18.4+0.502
Regression (r) 0.999 0.998 - 0.9860 0.999 0.999
Probability (p) < 0.001% < 0.001% < 0.001% 0.003% < 0.001%
SSRes* 0.511 0.366 0.363 0.357 0.361
*SSRes calculated for fit of untransformed data to Michaelis-Menten equation.

14 -- (a) (b)
n 0.5 --
13 -- i s s • Velocity Data Points
12 -- s~"
Michaelis-Menten Least
-- Squares Fit
11 0.4 -- _ _ Double Reciprocal Fit
10 -- v u
9 -- •

.~>. 8- • /" ~,03- /

o
2 7--
/ ,~

6- /
I
0.2
/,
5 -- •¢" • Velocity Data Points
B f•
] ~ Miehaelis-Menten Least Squares Fit ~•
4 1

• - -- D o u b l e R e c i p r o c a l F i t 4,•
3 --/ _ •t•
2 "F 0.1 i f m r

I [ I I I I I
j I I i I I I
0 20 40 150 80 100 120 -0.04-0.02 0 0.02 0.04 0.06 0.08 0.10 0.12

Substrate 1]Substrate

Figure 2a presents Data Set No 2 plotted as V vs [S]. The best fit (the non-linear least squares rio is shown as a solid line and the
worst fit (the double reciprocal linear regression) is shown as a dotted line. Figure 2b presents Data Set No 2 plotted as 1/V vs
1/[S]. On both graphs the two fits using the two methods are not readily distinguishable

BIOCHEMICAL EDUCATION 24(4) 1996

202

using evenly spaced data points and inverse transforma-

Table 5 Sample Data No 3 for Comparison of Different Methods tion of the data. On Fig 3b most of the data points are
of Estimating Km and Vinos.A set of sample data from Tayyab and crowded near the origin and a linear regression fit will be
Qamar. 35 This data set is from an experiment designed for teaching heavily biased by a few measurements at low substrate
elementary enzyme kinetics using the action of papain on a milk concentrations. The double reciprocal plot puts too much
casein as an example. The approximate position of the K., is shown emphasis on the small number of velocities measured at
by an arrow
low substrate concentrations (Table 6, Fig 3b). The
Substrate [S] Velocity double reciprocal regression fit is very clearly biased by
9.5 1.72
the velocity observed at the lowest substrate concentra-
19 3.91 tion (dotted line, Fig 3b) leading to a poor fit at higher
substrate concentrations and an overestimation of the Km
28.5 5.20
38 5.28
(Figs 3a and 3b). The SSRes of the unweighted double
57 7.75 reciprocal fit was 11.5 but weighting the data reduced the
85.5 7.61 SSRes to about 4. Both weighted and unweighted double
95 7.50
114.5 8.03
reciprocal fits were considerably worse than the other
133 7.93 fitting methods. Deviations from the fitted line, using all
171.5 7.70 the fitting methods, were not significantly correlated with
190 8.465 the velocity ( V i ) . 17'25'26

12 - - ( a ) (b)
0.6-- • Velocity Data Points • /
11
Michaelis-Menten Least /
10 s Squares Fit /
0.5 - - Double Reciprocal F i t / /
9 /
/
8 / /
0.4 --
7 ///

~ 6 "~ 0,3 /
/
5
,,y
4
.//'! -

- -
• V e l o c i t y Data P o i n t s
Michaelis-Menten Least
S q u a r e s Fit
D o u b l e R e c i p r o c a l Fit
0.2

LN
/t

I I I I I I i I I i I I I I I
25 50 75 100 125 150 175 200 -0.04 -0.02 0 0.02. 0.04 0.06 0.08 0.10 0.12

Substrate I/Substrate

Figures 3a and 3b present Data Set No 3plotted as Vvs [S] and 1/Vvs 1/[S] respectively. The best fit (the non-linear least squares
fit) is shown as a solid line and the worst fit (the double reciprocal linear regression) as a dotted line. The double reciprocal linear
regression gives over-estimations of Vat higher substrate concentrations (Figure 3a) and appears to be biased by a single data pair
(Figure 3b). Figure 3b shows the crowding effect of the inverse transformation on evenly spaced substrate concentrations and the
biasing effect of an atypically low velocity measured at the lowest substrate concentration

Table 6 Unweighted and Weighted Double Reciprocal and Eadie-Hofstee Plots Compared to a Non-Linear Least Squares Michaelis-
Menten Fit. Comparison of estimates of Kin and Vm~ using different fitting techniques using the data set shown in Table 5
L i n e w e a v e r - B u r k Fit H a n e s - W o o l f Fit E a d i e - H o f s t e e Fit Least Squares W e i g h t e d Inverse T r a n s f o r m s
M i c h a e l i s - M e n t e n Fit Wilkinson's V~4 Weighting
(NB This weighting is not valid)

K~ + SE 57.4 + 10.0 27.6 + 5.01 30.8 + 6.42 27.0 + 5.16 23.2 + 4.26
Vm.x + SE 13.00 + 2.07 9.57 _++0.446 9.9l + 0.805 9.70 + 0.505 9.52 + 0.508
Regression (r) 0.978 0.9904 - 0.8479 0.969 0.884
Probability (p) <0.001% <0.001% <0.097% <0.001% 0.03%
SSRes* 11.48 2.96 3.02 2.80 3.16

*SSRes calculated for fit of u n t r a n s f o r m e d data to M i c h a e l i s - M e n t e n equation.

BIOCHEMICAL EDUCATION 24(4) 1996

 203

Non-parametric methods are unaffected by single or a
few outliers. Non-parametric estimates of the median Km
and Vmax [Km=33.7 (-7.95, +21.1); Vmax=9.72 (--0.46,
+0.45)] are similar to estimates made using the para-
metric methods but indicate that Km cannot be estimated
very reliably. Here the use of a non-parametric method
alerts the user to the presence of aberrant data points,
resulting in biased parametric estimates of parameters.
Are the data sets suitable for the use o f parametric
statistics ?
If a data set is small (say n < 40) it is often difficult to
determine if it is suitable for analysis using parametric
statistics. 17'25'26 In our recent simulation studyTM we have
shown that the number of data points and the spread of
the points relative to the K~ can have large effects on
whether estimates of Km are normally distributed and
hence whether the use of parametric statistics is justifi-
able. The general conclusion drawn was that more data
points are needed than is generally realized (n > 10).
The Monte-Carlo simulator used in our theoretical
studyTM can be used to help design better experiments to
make improved estimates of Km and Vmax-For example, a
preliminary experiment can provide provisional estimates
of Km and Vm~ for a given number of data points, spread
of substrate values and error variance. An illuminating
way of using the Monte-Carlo Simulator is to use it to try
out alternative experimental designs that might provide
better estimates of these parameters. For example would
more be gained from (a) using more data points or (b)
using an alternative spacing of chosen substrate values?
squares) statistics to estimate the error-bars of the Km and
Vm.x was not really justifiable. An improved experiment
needs to be designed. The distribution is biased to lower
values and is flat in the region of the mean. A logarithmic
transform of the Km simulation data showed that it was
also inconsistent with a log-normal distribution
(p < 0.01).
Let us suppose that the experimenters had used dupli-
cates (n = 12). Given the estimates of Km, Vmax and the
variance would twelve data points have been sufficient? A
simulation run showed that Km would not have been nor-
mally distributed if 12 data points had been used
(D = 0.0309, 0.01 <p <0.05). Another simulation showed
that triplicates (n = 18) were necessary for Ko, to have a
distribution consistent with a normal distribution
(D = 0.0228, p > 0.05).
Perhaps an alternative arrangement of the substrate
values would have improved the statistical properties of
K m .1'2'15'16'23 The substrate values for Data Set No 1 were
arranged in an inversely proportional manner. 1'2 Simula-
tions using substrates, covering a similar range, but
arranged arithmetically and logarithmically were run.
Estimates of Km were still not normally distributed
(p<0.01) based on the Kolmogorov test. Thus, in this
case, the number of data points was more crucial than the
arrangement of the substrate values in achieving normally
distributed estimates of Kin.
1so-
Frequency Distribution of K m, D e t e r m i n e d from
Non-linear least s qua r e s fits, Compared to the Frequency
Distribution
and
of a N o r m a l
Variance.
Curve with S a m e Mean

The statistical properties of a large population

(n = 1000) of experiments with given estimates of the
100 ~ / .....
fitted parameters and a normally distributed variance can
be analysed. Each experiment will generate a least squares
estimate of Km and Vm,x.These are stored in a file and the " iiiii iiii
statistical behaviour of the population of estimates of Km
(and/or Vmax) can be analyzed. A conclusion can then be
drawn about the expected behaviour of an actual experi-
. . . . !,tl i !i !i i
ment that had the specified design and normally distri- 0 , , i;i!il iiiiiiiiiiii ii!iii
buted error using the non-parametric Kolmogorov 0.46 0.5 0,54 0,58 0,62 0.aS 0.7 0.74

t e s t . 17'18'25'26'43 Alternative arrangements of substrate values D a ta : Fitted Km [] Frequency

Velocity variance Normal Frequency
and different numbers of data points can be tried out. Evenly spaced data
= 184
pofnts
x 10" 6 •
CHI 2 : 86, df = 13, p << 0.001
n = 1000
Other error structures can also be tested, for example data
with a normally distributed relative error. TM Simulations Figure 4 Testing the statistical validity of the experimental
might show that a particular technique has an error vari- design used to obtain the data shown in Data Set No 1 using a
ance that is too high for there to be any hope of making Monte Carlo simulator. Frequency distribution of K,,~ deter-
reasonable estimates of Kin. mined from a non-linear least squares fit to the Michaelis-
Data Set No 1 used only six pairs of data points and so Menten equation, compared to a normal distribution of equal
we had serious doubt that the data set was really suitable mean and variance. 1000 Monte Carlo simulations were run
for the use of parametric statistics. TM For the given esti- on Data Set No 1 using the original set of substrate concentra-
mates of Km, V .... variance and spread of data points tions (n =6) and the estimated variance based on the non-
(Table 1 and Table 2), the Kolmogorov test on 1000 simu- linear least squares fit. The error of the data points used in the
lations shows that estimates of Km did not have a distribu- simulations was taken to be independent of substrate concen-
tion consistent with a normal distribution (D=0.0504, tration. Analyses using CHI 2 and the Kolmogorov test show
p <0.01). Fig 4 shows that the frequency distribution of that estimates of K,, based upon Data Set No 1 are not nor-
the Km estimates, found in the simulation is clearly not mally distributed and hence parametric statistics are not
normally distributed. Thus the use of parametric (least appropriate for Data Set No 1

BIOCHEMICAL EDUCATION 24(4) 1996

204
Data Set No 2 has 10 data points and a very good fit
using all the fitting methods discussed in the present
paper. From our theoretical study, it appeared that a suffi-
cient number of data points had been used TM. For the given
estimates of Kin, Vmax,variance and spread of data points
(Table 3 and Table 4), the Kolmogorov test on 1000 simu-
lations showed that K~ had a distribution consistent with
a normal distribution (D = 0.0271,p > 0.05). Thus, the use
of parametric (least squares) statistics to estimate the
error of the Km was justifiable.
Data Set No 3 has eleven pairs of data points and so
might have been expected to be suitable for the use of
parametric statistics28 For the given estimates of Kin, Vma~,
variance and spread of data points (Table 5 and Table 6),
the Kolmogorov test shows that estimates of Km were not
normally distributed (D=0.0547, p<0.01). If the data
points had been duplicated (n = 22), the distribution Km
would still have deviated from a normal distribution
(D=0.0349, p<0.01). It appears that triplicates would
have been necessary (n = 33) for Km to be normally distri-
buted (D = 0.0207, p > 0.05).
Data Set No 3 appears to have too many data points
well above the Km. A simulation experiment with substrate
values from 5 to 55 was run. The distribution of Km was
still not normally distributed (p < 0.01). The arrangement
of the chosen substrate values was less critical than the
number of data points employed. This conclusion agrees
with our previous theoretical study? 8 Data Set No 3 has an
error variance that is is too high for making good esti-
mates of Km.
Discussion
The three sets of data used in this paper are examples of
a data set spread approximately about the Km (Data Set
No 1), a set of data spread below the Km (Data Set No 2)
and a data set spread above the I ~ (Data Set No 3). The
non-linear fitting to the Michaelis-Menten curve gave
superior estimates of Km and Vm= using all three sets of
data, but the use of parametric fitting methods appears
only to be justifiable for Data Set No 2. Simulation studies
showed that that Data Sets No 1 and No 3 were not really
suited to using parametric statistics to estimate Km and the
non-parametric estimate of Km was the only valid estimate
of Km. The error variance was too high for the number of
data points used. Many more data points were needed.
The double reciprocal plot places undue emphasis on
enzyme velocities observed at low substrate concentra-
tions, where the relative errors of experimental data are
likely to be greatest, and assumes that the errors of 1/V,
rather than the errors of V are normally distributed? 'z'4"5'15
The double reciprocal plot gives both biased and indirect
estimates of K~ and Vr, a~.4
The double reciprocal plot gives estimates of Km and
Vmaxthat are both biased and imprecise. The double recip-
rocal method is consistently the worst method of esti-
mating K~ and Vmax values even using data from an
BIOCHEMICAL EDUCATION 24(4) 1996
experiment in which the IS] values were chosen so that
1/[Si] was as evenly spaced as practicable (Data Set No 2,
Table 3), as recommended by Cleland.l'2 It is a particularly
bad method to use if the substrate values are evenly
spaced (Data Set No 3, Table 5). The use of the
unweighted double reciprocal plot makes it difficult to
identify differences in experimentally determined Km and
Vm~ from one experiment to another and makes it par-
ticularly difficult to identify, and distinguish between,
competitive and non-competitive inhibition. It is
important to note in Tables 2, 4 and 6 that the correlation
coefficient can be very close to unity (indicating a very
good fit) and yet the errors of the estimated K~ and Vm,x
can still be large, of the order of _+50%.
The bias of double reciprocal plots is the more intract-
able problem because the errors of Km and Vm,x, deter-
mined using such plots, do not have even an
approximately normal distribution. 4'18 Poor precision can
be improved by doing several separate experimental
determinations and then calculating the overall mean I ~
and Vma~ values. 3 Bias cannot be eliminated by simply
doing more experiments: the mean of several biased esti-
mates of Km will be a biased overall mean.
Nevertheless, experimental data often fits double recip-
rocal plots very well but Dowd and Riggs 4 pointed out that
double reciprocal plots were highly deceptive, "We are
thus confronted with the paradox of obtaining the best
estimates from the 'worst fitting' line, and the worst esti-
mates from the 'best fitting' line! The undeserved popu-
larity of the Lineweaver-Burk method may well be based
upon just this ability to provide what seems a good fit even
when the experimental data are poor".
The estimates of the standard errors of K~ and Vm~
made from the double reciprocal plot is only approximate
and considerably worse than those made using the
non-linear least squares Michaelis-Menten fits (Tables 2,
4 and 6). The H a n e s - W o o l f plot is markedly better than
the unweighted double reciprocal method. A clear dis-
advantage of the method is that the same cumbersome,
and only approximate calculations, are used to convert the
intercept (Km/Vm,~) and slope (1/Vmax) into final estimates
of Km and Vm,x. Having the independent variable on both
sides of the Hanes-Woolf equation (eqn 3) does not pose
as serious statistical problems as having the dependent
variable on both sides of the Eadie-Hofstee equation
(eqn 4). Nevertheless, estimates of the errors of Km and
Vm~ derived from a H a n e s - W o o l f plot appear to be mis-
leadingly optimistic.
The limitations of the double reciprocal plot are
claimed to be partially overcome using a weighted linear
regression. I'2'4'1°'31'32'44 The appropriate weighting for
double reciprocal plots to counteract the biasing effects of
large 1/V~ values, is approximately proportional to the
fourth power of the expected velocity values (E(V)4),
assuming uniformity of variance of the values for V
(cfl-4'10"-12'32'33). The commonly used Vi4 weighting (Wilk-
inson's Method) is no more than an ad hoc approximation.
Tables 2, 4 and 6 show that the weighted linear regression

m e t h o d gives better results than the unweighted double
reciprocal plot but not as good as the non linear least
squares method.
T h e value of weighted double reciprocal m e t h o d is
doubtful because they do not give as good results as the
non-linear least squares method. It turns out that it
requires a higher level of statistical sophistication. T h e r e
is a unique solution to fitting a simple unweighted linear
regression but a weighted regression requires an iterative
weighted least squares fitting method. This involves more
sophisticated programming than in the case of an
unweighted non-linear least squares fit. A statistical
package would need to be capable of a non linear least
squares fitting to be capable of a weighted linear least
squares fit.
The Hofstee plot gives better estimates of K~ and Vmax
than the double reciprocal m e t h o d but the standard for-
mulae for errors of slope m and y-intercept 25'26 used to
calculate the standard errors of Km and Vm~ are not
strictly valid. T h e r e is little to gain from the use of the
linear transforms of the M i c h a e l i s - M e n t e n equation
when the least-squares technique clearly gives the most
precise estimates of Km and V .... and a statistically rig-
orous m e t h o d of calculating their standard errors. The
H a n e s - W o o l f plot is probably the plot of choice, if a
linear plot is to be used for the visual inspection of experi-
mental results and for identifying differences between
experimental treatments.
Even though the dependent variable appears on both
sides of the E a d i e - H o f s t e e equation (eqn 4), Dowd and
Riggs 4 claimed that computer simulations showed that the
error of K,, and Vm,~ closely approximated a normal distri-
bution and hence was a more appropriate method than the
double reciprocal plot. Ritchie 42 used the E a d i e - H o f s t e e
plot to estimate Km and Vm~x in each experiment. Several
sets of Km and Vmaxvalues obtained in separate experiments
were then used to calculate overall Km and Vmax values.
Most of the variance was between separate experiments and
so improvements in the standard errors of K~ and Vm,~
within each experiment would have had little effect on the
accuracy of the overall means.
Faulty experimental design can also lead to problems in
determining Km and Vm,~.~'~8 Dowd and Riggs 4 made a
survey of 28 papers in which Km and V .... values were deter-
mined: nine studies used spreads of substrate concentra-
tions in which all were below the Kin. At substrate
concentrations below the K,,, V is approximately propor-
tional to [S] and so the data may have so little curvature that
a Michaelis-Menten curve cannot be fitted to the data. 2~
Spurious fits to a Michaelis-Menten curve may be
encountered because the Michaelis-Menten model is
inappropriate. 2'3'3°'38'3'~ One example of a case where the
Michaelis-Menten model might be applied inappropriately
is where velocity is simply directly proportional to substrate
concentration (V= C[S]). The data shown in Table 3 (Data
Set No 2) was sufficiently accurate for good estimates of Kn,
to be made even though most of the data points were below
the K.,.
BIOCHEMICAL EDUCATION 24(4) 1996
205
We will briefly deal with two approaches to improve
estimates of Km and Vmax: (a) allowing for a non-zero velo-
city at zero substrate as discussed by Selwyn31 and (b)
assuming a constant relative error, rather than a constant
error. The model for a Michaelis-Menten curve with a
constant background rate is,
[ S l V .....
v - - (lo)
[S] + Km
where, V, [S], Vmax and Km have their previously defined
meanings and C is a constant background velocity that
occurs even in zero substrate. It is much more difficult to
estimate three parameters with precision than only two.
Only a non-linear least squares fit can be made to such a
model. A fit was made to all the sets of data used in the
present study. In all three cases, C was not significantly
different from zero and so there is no evidence for invoking
this more complex model. The errors in estimates of Km and
V ..... were much larger using eqn (10). The poor fits to the
Michaelis-Menten relationship found for Data Sets No 1
and No 3 cannot be accounted for by postulating a 'blank' or
'background' velocity.
In all three data sets, the velocities have a range of over
a factor of 5. It is perhaps more realistic to assume a con-
stant relative error [error of V(i) is proportional to V(i)].
The easiest method for allowing for a constant relative error
is to log-transform both sides of eqn (1) and then use a non-
linear least squares fit,
Ln(V) = Ln([S]) + Ln(Vm~,x)- Ln([S] + Kin) (11)
The log-normal least squares model did not improve the
fits to any of the data sets used in the present study and the
estimates of Km and V ..... were not significantly different
from those obtained using the non-linear least squares fit
with a normally distributed error structure (eqn 1). Plots of
residuals showed no tendency for them to increase as V
increased 17. The constant relative error model should only
be invoked if there is some evidence in favour of it.
It is worthwhile to compare the results of the present
study using parametric statistics to estimates of Km and Vm,x
obtained using non-parametric methods similar to those
promoted by Cornish-Bowden? ~'~z'~ 24 Taking into account
the approximate 95% range limits of the median estimates
of Km and V,,,~x 25'2~' we have shown that non-parametric esti-
mates of Km and Vm~xare consistently similar to the para-
metric least squares Michaelis-Menten fit. The
non-parametric method is a useful technique for checking
parametric estimates of Km and V .... that might be biased by
one or a few pairs of aberrant data. Our use of a Monte-
Carlo simulator ~ shows that it is not really justifiable to
quote parametric estimates of either K~ or V ..... for Data
Sets No 1 or No 3.
Serious questions must be asked about the statistical
validity of many K~ values in the literature. To gain an
impression of techniques currently used by experimental
biochemists, Ritchie and Prvan 's made a survey of recent
volumes of the B i o c h e r n i c a l J o u r n a l (1993, Volumes 289 and
206
290). The average number of data points in any one experi-
ment was 6.1 + 1.2 (range 3-13, n =20) but many papers did
not mention how many data points they used. Our simula-
tion studies showed that more than 10 data points were
needed for estimates of Km to be normally distributed. TM
Users of non-linear Michaelis-Menten fitting methods do
not use more data points than users of double reciprocal
methods. TM Any gains made using better fitting methods
were therefore marginal. It appears that many biochemists
are currently using statistically unsatisfactory methods for
measuring enzyme kinetic parameters: there are three
underlying problems, often in combination: (i) unsatisfac-
tory, obsolete, fitting methods for routine work, (ii) too few
data points and (iii) poor choice of sets of substrate
concentrations? s
The problem of too few data points (eg Sample Data No
1) proves to be a pervasive problem, simply solved by
routinely using more data points. Poor choice of the set of
substrate concentrations is a more difficult problem (for
example Sample Data No 1 and No 3). The spread of sets of
substrates concentrations chosen by the experimenter can
have large effects on the statistical properties of Kin. A data
set can appear to have a sufficient number of data points
and a very good correlation (r) and yet Monte Carlo simula-
tion methods ~8might show that the data are not suitable for
parametric statistical analysis. The Monte Carlo simulator
thus provides a useful tool for assessing preliminary data
sets and the experimental design used.
The I ~ and Vmax of Michaelis-Menten type processes
are very important to many branches of biology, but often
no statistical information is available on their precision.
More attention should be paid to the minimization of bias
in Km and V .... and the importance of their statistical errors
and the error structure of biochemical data 44. Many Km and
and Vm,x values in the literature are likely to be biased,
inaccurate and imprecise. Commonly accepted cases of
competitive and non-competitive inhibition might not be as
well established as usually thought
Acknowledgements
The authors wish to thank Professor N A Walker (Univer-
sity of Sydney) for providing a BASIC version of Colqu-
houn's 'Pattern search' ALGOL 60 program. One of the
authors (R JR) is an Australian Research Fellow and
gratefully acknowledges his funding from the Australian
Research Council. Professor Walker and Professor A W
D Larkum (Sydney) encouraged this work and made
helpful comments on the manuscript. Dr S Tayyab (Ali-
gargh Muslim University, India) kindly furnished us with a
table of their original data. 35
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