22 I N I T I A L RATE M E T H O D S [2]

Concluding Remarks
The development of a reliable initial-rate assay may appear to be an in-
surmountable task involving the interplay of many variables. Yet, for
those interested in enzymology, this activity frequently presents the
opportunity to uncover new aspects of the behavior of a particular en-
zyme. Many fascinating details of enzyme mechanism and metabolic reg-
ulation have evolved from such exercises, and a valid assay represents a
powerful tool to probe further.

[2] T e c h n i q u e s in C o u p l e d Enzyme Assays

By FREDERICK B. RUDOLPH, BENNETT W. BAUGHER,
a n d R O B E R T S. B E I S S N E R

The major problem in initial-rate kinetic studies is often the method of

assay. If possible, an assay should be accurate, sensitive, and convenient.
In addition, the ability to continuously monitor a reaction process is of
great value. Unfortunately, many reactions do not produce changes in the
spectral or other properties of the reactants and cannot be directly mea-
sured. To allow continuous assay of such reactions, the formation of a
product can be measured by addition of an auxiliary enzyme that pro-
duces a measurable change. Such methods are sensitive and convenient,
but have certain disadvantages, the most important being a lag period be-
fore the steady state is reached, which will be detailed in this chapter.
Various theoretical analyses of the use of consecutive enzyme reactions
for assay systems have been made and will be considered here along with
practical aspects of their use, precautions to be observed, and examples
of such assays.

Theory
Models and Analysis of Coupled Systems
A number of approaches describing ways to ensure valid coupled
assays have appeared in recent years. 1-8 The basic problem is to deter-

1 H. U. Bergmeyer, Biochem. Z. 324, 408 (1953).

H. Gutfreund, " A n Introduction to the Study of E n z y m e s , " pp. 302-306. Blackwell, Ox-
ford, 1965.
a W. R. McClure, Biochemistry 8, 2782 (1969).
4 C. J. Barwell and B. Hess, Hoppe-Seyler's Z. Physiol. Chem. 351, 1531 (1970).

Copyright O 1979by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 63 All rights of reproduction in any formreserved.
ISBN 0-12-181963-9
[2] COUPLED ENZYME ASSAYS 23

mine that auxiliary enzymes will react at a rate that allows monitoring
only o f the steady-state concentration of the product(s) (P) of the reaction
being studied, not of the rates of the auxiliary enzymes. The systems have
an inherent lag time prior to the steady state that must be analyzed and
minimized. The simplest example of such a system is
A , P , Q (1)
El E,

where E1 is the e n z y m e whose activity is being measured (primary en-

zyme), E2 is the auxiliary enzyme, and kl and ks are the rate constants for
the respective enzymes. The general approach to such assays has been to
use a large excess of the auxiliary enzymes to assure steady-state condi-
tions. The behavior of such a system was first treated quantitatively by
McClure 3 using the following assumptions. (1) k~ is the rate constant for
E~, which is assumed to be an irreversible zero-order step. To meet this
criterion, all substrates for E1 must be saturating or only a small fraction
of the substrates can react. Irreversibility is assumed since P is continu-
ously removed by E~ during the assay. (2) The second reaction is irrevers-
ible and first order with respect to P (rate constant ks). This necessitates
that P ~ Kp (the Michaelis constant for P for Ez) and that the other sub-
strates for E~ be nearly saturating. If the equilibrium for the reaction cata-
lyzed by E2 lies to the right or only a small amount of reaction occurs so
that the other substrates for E2 are not depleted, irreversibility can be as-
sumed. Proper choice of the auxiliary e n z y m e will satisfy this condition.
With these assumptions, it is possible to calculate the amount of E2 re-
quired for a theoretically correct assay. For the sequence in Eq. (1)
dP/dt = kl - kzP (2)
which integrates, using the limits t = 0 ~ t and P = 0 ~ P, to
P = (kl/kz)(1 - e -I'~t) (3)
as t --~ 2, p approaches its steady-state concentration (Pss) or
Ps~ = k l / k 2 (4)
Equation (3) can be rearranged to
In[1 - k 2 P / k l ] = - k 2 t (5)

5 B. H e s s and B. W u r s t e r , FEBS Lett. 9, 73, (1970).

J. E a s t e r b y , Biochim. Biophys. Acta 293, 552 (1973).
7 p. W. Kuchel, L. W. Nichol, and P. D. Jeffery, J. Theor. Biol. 48, 39 (1974).
8 A. Storer and A. C o r n i s h - B o w d e n , Biochem. J. 141,205 (1974).
24 INITIAL RATE METHODS [2]

These equations allow calculation of the amount of Ez required in an

assay to reach the steady state for P (P,~s) in a given period of time. Com-
bining Eqs. (4) and (5) results in
In(1 - P/P,~s) = -k2 t (6)
If Kp >> P~.~as assumed initially, then, from the M i c h a e l i s - M e n t e n equa-
tion for E2,
V2Pss V2Pss
V 2 - K p + Ps~ Kp - k2Ps~ (7)

This expression can be substituted into Eq. (6) to give an expression for
the amount o f E2 (or Vz which is the maximal velocity for E2) required to
attain a given fraction o f the steady-state phase of the coupled reaction at
any given time t.
-ln(1 - P/P~0 Kp
V2 = (8)
t

If Fp is the fraction of Pss desired at time t, the equation can be expressed

as

- 2 . 3 0 3 log (1 - Fp) Kp
V~ = (9)
t

The time required to each Fp (t*) can be calculated from

- ln(l - Fp)
t* - (10)
ks
To use expression (9), one decides on the F~ desired with a given lag
period and calculates Vs. Only K v has to be known to make the calcula-
tion.
Certain features of the Eqs. (7)-(9) are apparenta: (1) k2 is influenced
by the ratio V2/Kp and is not just dependent on a large excess of E2; this
point is illustrated in Fig. 1. Both the e n z y m e concentration and Kp will
influence the lag time. Either an increase in Kp or decrease in E2 will cause
an increase in the lag time. These factors should be considered if iso-
zymes or similar e n z y m e s are available for the same assay. (2) The time
required for establishment of the steady state (Ps0 is independent of ki.
The system is therefore suitable for assaying any activity of the primary
e n z y m e as long as P~ ~ Kp. This is an important factor and suggests that
a lower limit for Kp exists and should be considered when a choice of aux-
iliary enzymes is made. (3) Pss is a function of both kl and ks.
The attainment of the steady-state concentration of P is shown in Fig.
2. In this hypothetical example sufficient E~ is present to give the steady
[2] COUPLED ENZYME ASSAYS 25

1.0

0.5

005 0.10

Kp

FIG. 1. A plot of amount of auxiliary enzyme, E2, versus the Ko for various indicated lag
times (t*)

o
E01/
[P]

I I I
5 I0 15 20 25 50
Time (sec)

FIG. 2. A plot of the concentration of the intermediate P, product, and the final product,
Q, as a function of time.
26 I N I T I A L RATE M E T H O D S [2]

state within 5 sec, after which the formation of Q is constant ( d Q / d t = C

and - d P / d t = 0) and initial velocity conditions are achieved.
Assay systems utilizing more than one auxiliary enzyme are more dif-
ficult to analyze. For the system
A , P , Q , R (1 1)
E~ E2 E3

where E1 is the primary enzyme and Ez and E3 are the auxiliary enzymes,
McClure has applied an analysis similar to that described above for Eq.
(1). An expression for [Q] analogous to Eq. (3) is

Q _ kl k~ [e -k~t - ( k z / k s ) e -k3t] (12)

ks ks k2

This equation contains the following features: (1) The first term is equal to
the steady-state concentration of Q (Q~0, and the negative term is the time
dependence of steady-state attainment. (2) The rate of steady-state attain-
ment is symmetrical with respect 1~okz and k3, allowing values of kz and k3
to be interchanged without affecting the lag time. (3) The time required to
achieve a given fraction of Qs~ is not a function of kl.
Equation (12) cannot be solved as easily for t or t* as the two-enzyme
case, but some conclusions were drawn by McClure? If either k2 or k3 is
large compared to the other rate, then the lag time is primarily dependent
on the smaller value. The equation reduces to

t* - -ln(1 - FQ) if ks >> k2 (13)

k2

or

t* - -ln(1 - FQ) if k2 >> k3 (14)

k3
In fact, the difference between the two constants need only be 4- to 5-fold
for satisfactory prediction of t*. When neither auxiliary enzyme is in
excess, there is no single solution for t*. McClure 3 has solved Eq. (12) nu-
merically, and the results are presented in Fig. 3 as nomograms showing
the time required to reach 99% of Qs~ for different values of k2 and k3.
A similar approach to dealing with the lag time of a coupled assay has
been presented by Easterby,6 and his approach allows calculation of some
useful values. A resultant equation from his treatment of Eq. (1) is
[P] = vl (t + r e -t/¢ - "r) (15)
where r, the transient (lag) time = K p / V 2 and Ps~ = vaT. For this treat-
ment the plot of Q formation versus t is shown in Fig. 4. The P~s concen-
[2] COUPLED ENZYME ASSAYS 27

005

0.04 "k
2-- Vm°xEz /
>(
NO.07 i I i i i
0
w
0.06
0.03
0 ~ o.os
E
0.02
~- 0.04
¢-.
0.03
.w
E 0.02
v 0.01
T~
0.01
T N
se,cI se,c IS?:l
0 5 10 15 o o.o2 o.o, o.o6

Time to reoch 0-99[S]ss(Sec)LJ k~ (min) or Kmo / VmaxE 3

(o) (b)

FIG. 3. Nomograms of McClure showing the time required for 99% attainment of the
steady state (t*) in a coupled assay using the auxiliary enzymes. Rz and R3 represent the
first-order rate constants (Wmax/Km)for the two coupling enzymes. Adapted with permission
of W. R. McClure and the American Chemical Society (copyright holder), from Biochemis-
try 8, 2782 (1969).

Time

FIG. 4. A plot of the formation of the final product Q, of E2 as a function of time. The x in-
tercept represents the transient time (z) as discussed in the text, and the y intercept repre-
sents the steady-state concentration of intermediate P (Psi).
28 INITIAL RATE METHODS [2]

tration is found from the y intercept, and the transient time (z) from the x
intercept. This information allows calculation of vl, the velocity of the
reaction being studied. Determination of vl is done at a series of A con-
centrations, and the velocity dependence on substrate concentration is
plotted. The transient time approximates Kp/V2 only if V2/Kp ~> V I / K A .
The ability to determine P~ allows a check on the assumptions used in
McClure's derivations. If P.~s < Kp, then the methods o f calculation of the
amount of coupling e n z y m e s required are more valid. It also allows moni-
toring if product inhibition is a potential problem in the assay.
Easterby 6 has also treated assays using two or more auxiliary en-
zymes. He found the transient time for reaching the steady state for the
assay to be the sum of the transients for the individual coupling enzymes.
Thus, the lag time increases as the number of auxiliary enzymes in-
creases. Of importance is that this treatment also indicates that the tran-
sient time is independent of the initial rate-limiting enzyme. Changing the
activity o f E1 does not change the lag. Easterby 6 also showed that the min-
imum time for accurate measurement of initial velocity (steady-state
intermediate) has to be at least five times the transient time.
Storer and Cornish-Bowden 8 have considered coupled assays without
assuming that the second and subsequent reactions followed first-order
kinetics, as was done by McClure and Easterby. This more general treat-
ment for the reaction sequence
A ~' ) P v2 ) Q (16)

where Vl and v2 are the experimental velocities, gives an equation for the
time (transient time) required for v~ to reach any given value as
t* = c~Ko/vl (17)
where d~ is defined as
V2v~
- (V2- Vx)2 In [vl(V2- v2)] vlv2
LV~(vl- ~ (v2- v2) (v2- Vl) (18)
is a dimensionless number and a function of the ratios v2/vl and vi/Vz
only. V2 is the maximal velocity of the auxiliary enzyme. Values o f 6 can
be calculated at various values o f vl/V2 and v2/v~and are given in Table I.
The value of v~/vl depends on the accuracy desired. In a precise assay
it is desirable that it be at least 0.99. The amount o f E2 required for a de-
sired accuracy can be estimated for a given vi and the lag time calculated
from Eq. (17).
The analysis of Storer and Cornish-Bowden s also considered multiple
coupled systems. They found that, by treating each step individually, the
lag time was bounded by t~ . . . tn < hag < £/'=~tl. That is, it was longer
than any individual time but less than the sum of all. Assuming that the lag
[2] COUPLED ENZYME ASSAYS 29

TABLE I
ANALYSIS OF LAG TIMES FOR COUPLED ASSAYSa'b

vl/V2 vl/v2 = 0.95 0.99

0 0 0
0.05 0.16 0.25
0.10 0.35 0.54
0.15 0.56 0.89
0.20 0.81 1.31
0.25 1.11 1.81
0.30 1.46 2.42
0.35 1.88 3.18
0.40 2.39 4.12
0.45 3.02 5.32
0.50 3.80 6.86
0.55 4.79 8.91
0.60 6.08 11.7
0.65 7.80 15.6
0.70 10.2 21.4
0.75 13.6 30.3
0.80 18.7 45.5
0.85 27.2 74.3
0.90 42.8 141
0.95 77.9 377

Adapted from A. Storer and A. Cornish-Bowden, Biochem. J. 141,205 (1974).

b The time required for v2 (for the auxiliary enzyme) to reach the specified percentage
of the steady-state velocity (vl) of the primary enzyme (E0 is given by t = q~Kp/v~.

time is the sum gives a useful upper limit similar to the method of Eas-
terby. 6 The experimental study by Storer and Cornish-Bowden s illus-
trated that linearity of an assay cannot be assumed unless the apparently
linear portion is ten times as long as the obvious lag period.
The techniques discussed so far have assumed that the steady state
reaction of the primary e n z y m e can be measured with accuracy. The lag
period has been described only as to its duration and how to shorten it.
Kuchel et al.r have solved the basic equations describing Eq. (1) by for-
mulation as a set o f Maclaurin polynomials. This treatment leads to the
following equation
V1V2 Ao t 2
[P] = 2Kp (KA + Ao) (19)

where Ao is the initial A concentration.

30 INITIAL RATE METHODS [2]

A plot of P versus t z gives an initially linear plot with a slope

V2/2Kp • V1Ao/(KA + A0), which describes a rectangular hyperbola. The
slopes o f a number of A0 concentrations are plotted in double reciprocal
form to give an x intercept of - 1 / K A and a y intercept o f 2Kv/V1Vz. Prior
analysis of the V2 and Ko allows calculation o f both V1 and KA. This analy-
sis can be extended to any number of auxiliary enzymes by similar plots.r
This treatment will apply best when the activities of the primary and aux-
iliary enzymes are similar. A long lag time is needed to obtain the initial
linear slope of the P versus t ~ plot. If the lag is short, the data are readily
analyzed by the techniques described by McClure 3, E a s t e r b y f and Storer
and Cornish-Bowden. s This technique is of use particularly if the coupling
enzymes are not available in sufficient quantities to saturate the system
and does avoid some difficulties inherent in coupled systems. Additional
information concerning individual rate constants can be obtained from
pre-steady state studies as described by Roberts. 9 These are beyond the
scope and intent of this report but are available for reference.
The major conclusions of all the theoretical treatments on coupled en-
zyme assays are that they can be designed so that lag times are known and
minimal and the measured velocity accurately represents the steady-state
velocity of the primary enzyme. The calculations will usually provide the
correct answers, but certain precautions and limitations must be consid-
ered and will be discussed in the following sections.

Practical Aspects
The basic question in designing a coupled e n z y m e assay is how much
auxiliary enzyme(s) to add to ensure a short lag time and accurate mea-
surement of the e n z y m e being studied. It would be convenient if one
could simply say to use a 10-fold excess of the first coupling enzyme and
100-fold excess of the second e n z y m e if it is necessary, which has been a
convention among some enzymologists. H o w e v e r , the systems may not
always work well even with these excesses of coupling enzymes. The ex-
pense of the enzymes, contamination with other proteins, and other
factors necessitate a more defined approach to the problem. The theories
discussed above have the limitations that they are based on models re-
quiring certain assumptions but they generally are experimentally appli-
cable.
The choice of auxiliary enzymes is obviously dictated by the products
formed in the reaction. A number o f examples of coupled assays are listed

9 D. V. Roberts, "Enzyme Kinetics." Cambridge Univ. Press, London and New York,
1977.
[2] COUPLED ENZYME ASSAYS 31

in Tables II and III. This listing is not an exhaustive compilation of such

assays but generally represents recently developed methods. Included in
Table III are a number of stopped-time assays, in which auxiliary en-
zymes are present during the reaction to allow formation of an identifiable
product. The reactions are stopped at various time intervals, and the
product is measured. The use of the coupled system requires that the
assay times be done after the steady-state condition is achieved. 3 The ex-
cellent series entitled "Methods of Enzymatic Analysis" edited by H. U.
Bergmeyer TM should be the primary source for finding suitable systems for
coupled enzyme assays. The series details assays for both enzymes and
metabolites by using enzymic analysis. Such methods can be generally
adapted to kinetic assays. Choices between systems should be made by
comparing the Km's for the measured substrates and activity of the auxil-
iary enzymes. Different isozymes can be more useful in coupled systems
as illustrated by the fact that the H4 isozyme of lactate dehydrogenase is
much better than the M4 isozyme on coupling systems. 11
An interesting suggestion has been presented by Marco and Marco, ~z
who proposed using an alternative substrate, 3-acetyI-NAD, for dehy-
drogenase coupling enzymes. This analog has a more favorable equilib-
rium for 3-acetyl-NADH formation, allowing assays monitoring the pro-
duction of 3-acetyl-NADH to be done in the pH range of 7-8. Normal de-
hydrogenase assays are not as effective in this range. Also, the extinction
change is higher than for NADH so the assay is even more sensitive. In
general, alternative substrates can offer ways to develop better assays for
a given reaction. The number of coupling enzymes should be minimized,
but if the enzymes are readily available the sensitivity of the given assay
should be the most important factor. Spectrophotometric assays are nor-
mally sufficiently sensitive for use if an adequate change in extinction
coefficient is involved. A change of at least 0.05 to 0.1 OD unit is neces-
sary for good analytical measurement. For NADH, a change in concen-
tration of 8 p,M will generate an OD change of 0.05 in a 1-cm cuvette. If
the coupled assay contains a species that has a high absorbance, care
must be taken to avoid stray light errors, a3 A single-beam spectropho-
tometer is not accurate at an absorbance above 2 owing to stray light ef-
fects. Beer's law should be verified for the spectrophotometer being used
to ensure accurate measurements. Changes in substrate concentration or

10 H. U. Bergmeyer (ed.)"Methods of Enzymatic Analysis," 2nd ed., Vols. 1-4. Academic

Press, New York, 1974.
11 M. Chang and T. Chung, Clin. Chem. 21, (1975).
12 E. J. Marco and R. Marco, Anal. Biochem. 62, 472 (1974).
13 R. L. Cavalieri and H. Z. Sable, Anal. Biochem. 59, 122 (1974).
32 1N1TIAL RATE M E T H O D S [2]

6
6
~o
ta

¢11
e-
~ ~
~.~,~< o ~< r ~ ..-,~
< <

e.,

r. g

N o ,..1

~- ~. ~-~'~ o
Z ,v[. <

~u
a: := =a: =
<

9 < < < < << .~ ,~ ~

0 Z Z Z Z ZZ Zxa "~
"r. • <
0 .~ g z
8e-, ge..

,- g.
~= .g
e~ e-, ~I
r~ e.~
o E

ca
e..
E
~, ,- 0
°~
< < L) t=
[2] COUPLED ENZYME ASSAYS 33

g
X ~a
0

¢)

g
~_, ~ ~-~ ~ = -~ -= ~~ ~ ~
-~ = "a o "-
g

< < < a:

gg . 0a o g
gg g ~ = ,~ .o
a: "0 g
r'.,
,.,-.I
o

'a
o
"o ~
~.. ca "~ u ° ° ~a ~4 ,~ ~a u "0

~o~ o_,,~ o ~ o

N a:
e~ 0
a: ~ a: a: < < < =
z z ~ Z Z o
< < < < ~
zo = gz z z ~ z
,- .=_ ~ .r ,- e.,
= .~ .~
~o°.~
~" "~ ~ ~1~

o
g g .g <
g8 o = o 0a
o= ggg g ,.2.' .-=
>. ~ _o ¢II

< ~ o <
34 INITIAL RATE M E T H O D S [21

E
~ m

~ ~ -
~ -~ , "~~
•- =,~ ~ ~..~-
~ _~. ~ ~ ~
~ :~ ~,~, <

•~ ~ .~ .'~
>'~ ~ ~

<
[.-,

.~ == :=
< .~ ._~ < <
Z =, ~z z
"t- ~-'~, o ~
o ¢)

E
-t -,i

©© ~ "~ ._~ .~.

..od
~,4 ==~-," ~,~

"6

;= :=z
[2] COUPLED ENZYME ASSAYS 35

r~
o~

,..¢ e,~

r.- r.-

"~._ ~.~ ~ ~ ~ .~ ~ _~
p.
.,..~
~o

O', I, ~ D-
~ e-. tch r"-

Ca

m Ca

-s =. g,
=

• ¢-, e-' ~1 ;~

. m ~.a,Z N ~.® r~ .
C
e-

N N c.--=N ,= • . d ' ~ g
m
. o" m
~
¢~
= a.
.
=:
.--d
ai r ~ 4 ~ ~5
~ .'~

r.A~d < , , i ~= • .'- o

Z mm,~
 36 INITIAL RATE M E T H O D S [2]

-a -~

" ~ ~ o
t-.am

.~ - = "~ ~

, ..=
~ 1 ~.

<

< ~

.I
, ~ = ~ . .~ .~
D
0

© rL

Z
<
M
M

Z
.~ = ~ .= ~.~
~-
c~ "I~ 7~
'~ "~

,...?
<

C ~',~

.~ ~ ' ~ 0

~<~.~
>

"6

< Z
[2] C O U P L E D E N Z Y M E ASSAYS 37

addition of inhibitors that absorb at the assay wavelength can cause

serious errors and lead to incorrect interpretations. Fluorescence assays
are 2 - 3 orders of magnitude more sensitive than spectrophotometric
assays if a fluorescing species is involved. Self-quenching can be a
problem when disappearance of fluorescense is being measured. 14 Reac-
tions can be coupled to enzymes that release or take up protons and as-
sayed by use of a p H star. This method is probably not as sensitive as
spectral assays but would be useful in some cases. The use of isotopes in
coupled assays as illustrated in Table III can be very sensitive, limited
only be separation methods and the specific radioactivity of the substrate.
Once the choice of coupling system and method of assay is made, the
parameters of the auxiliary enzymes must be evaluated. The Vmax and Km
for each auxiliary e n z y m e should be determined with identical buffer,
ionic strength, temperature, and other conditions as will be used in the
actual measurements. It may be necessary to consider the inhibitory ef-
fects of substrates or inhibitors for each e n z y m e on the others. For ex-
ample, glucose-6-phosphate dehydrogenase is significantly inhibited by
free ATP, producing a long lag time 15 and limiting its use for measurement
of glucokinase and hexokinase activity in the presence o f high free ATP.
The measured kinetic parameters are likely to be different from manufac-
turer or literature values but are the proper values for the system being
studied. In addition, the solution in which the auxiliary enzyme is kept
must not cause inhibition of the primary enzyme. Ammonium sulfate is a
particular problem requiring dialysis of the auxiliary enzymes prior to
use. Even albumin, which is often added to stabilize proteins, can bind
many substrates and cofactors and cause inhibition in that manner. If pos-
sible, the coupling enzymes should be in the same buffer system as the
assay. Use of small molecular seive columns or rapid dialyzers will help
avoid stability problems encountered during normal dialysis.
The amount of auxiliary enzymes needed can then be calculated as
described either by McClure 3 or Storer and Cornish-Bowden. 8 The
parameters to be specified are the ratio of vz/vl or the fractional attain-
ment (Fp) o f the steady-state rate and the lag time before Fp is reached. To
ensure accurate assays, F o should usually be specified as 0.99 so that the
measured rate represents 99% of the steady-state rate of the primary en-
zyme. Lag time will depend on the system being studied. A lag of 30"sec,
or longer, may not be a problem in some assays if a long linear region is
then observed. If the primary e n z y m e is suspected of being hysteretic, TM

~4 K. Dalziel, Biochem. J. 80, 440 (1961).

15 G. Avigad, Proc. Natl. Acad. Sci. U. S. A. 56, 1543 (1966).
16 C. F d e d e n , J. Biol. Chem. 245, 5788 (1970).
38 I N I T I A L RATE M E T H O D S [2]

the lag time should be minimal to ensure actual m e a s u r e m e n t of the pri-

m a r y e n z y m e ' s behavior.
The two methods can be c o m p a r e d in the following examples. Storer
and Cornish-Bowden 8 used the assay of glucokinase with glucose-6-
p h o s p h a t e dehydrogenase for illustration of their method. The Km for glu-
cose 6-phosphate was found to be 0.11 m M under assay conditions. T h e y
specified that the lag time prior to v2 = 0.99 vl would be 1 rain and vl
would not exceed 0.04 m M min -a. Substitution of these values into Eq.
(17), t* = chK~/vl, gives 4) = 0.36. Using Table I and interpolating for
this value of ~b shows v~/V2 = 0.08 or V2 = 0.5 m M rain -1 or 0.5 I U / m l .
Using M c C l u r e ' s analysis with Eq. (9) gives

- 2 . 3 0 3 log (1 - Fp)K o
V2 = = - 2 . 3 0 3 log (1 - 0.99) (0.11)
t
= 0.51 m M rain -1 or 0.51 I U / m l
So, for a simple system, the two treatments will give similar answers. As
vl decreases, the amount of E2 added in the Storer and Cornish-Bowden 8
treatment will be s o m e w h a t less, but not proportionally since both ~b and
V2 are related to Vl.
An e x a m p l e of a two-auxiliary e n z y m e couple was presented by Mc-
Clure 3 for a generalized kinase assay using p y r u v a t e kinase and lactate de-
hydrogenase. The values used were kl = 0.05 m M rain -~, KAOP =
0.21 mM, and Kpy~uvate -- 0.14 mM. The transient time (t*) was chosen to
be 12 sec. F r o m the n o m o g r a m in Fig. 3(b), if t* is 12 sec then ks and k3
must be larger than 1/0.043 or 23 min -~. Assuming that k2 and k3 are
approximately equal and that sufficient values are kl = k3 = 33 min -1,
then k2 = V2/KADPand ks = V3/Kpyruvateand k2 = 6.9 I U / m l of p y r u v a t e
kinase and ks = 4.6 I U / m l of lactate dehydrogenase. Using equal
amounts of k2 and ks allows the minimum total units of e n z y m e to be
used?
Using the Storer and Cornish-Bowden 8 analysis necessitates separa-
tion of the transient time for each couple and calculation of the ~bfor each
reaction as was done in the one auxiliary e n z y m e case. If the tl and t~ are
a s s u m e d to each equal 6 sec, then the tb for p y r u v a t e kinase is equal to
(t*) (v0 (0.1) (0.05)
- - - - 0.024
Kp 0.21
The ratio of v~/V2 from Table I is 0.005, so V2 = 10 I U / m l . F o r lactate de-
h y d r o g e n a s e v3 is assumed -~ v2, so the calculation can be made. 4) =
(0.1)(0.05)/(0.14) = 0.036, and f r o m Table I v2/V3 = 0.007. Thus, the lac-
tate dehydrogenase concentration should be 7.1 I U / m l . The two methods
once again give similar results although McClures '3 m e t h o d is probably
[2] COUPLED ENZYME ASSAYS 39

easier to handle for a two-auxiliary enzyme system. The treatment of

Storer and Cornish-Bowden s gives high values for V2 and V3 because of
the assumption that the lag times are independent. The actual observed
lag time will be shorter than assumed in the calculations. The treatment of
Storer and Cornish-Bowden a has the advantage that it can be adapted to
3 or more coupled enzymes.
Calculations of transient time for a given assay using defined levels of
coupling enzymes can be done using the methods of McClure, 3 Easterby, 6
and Storer and Cornish-Bowden. s The analysis of Easterby 6 illustrated in
Fig. 4 allows easy determination of the lag time and the steady-state con-
centration of the intermediate product. Knowledge of that concentration
allows confirmation of the validity of the assumptions in the models pre-
sented in the Theory section and analysis of product inhibition effects by
the intermediate product.
Making the calculation as to the amount of enzyme to be added does
not free the investigator from making sure that the coupled assay really
measures the velocity of the primary enzyme. Test assays should be run
with levels of the coupling enzymes 2- to 10-fold higher than calculated,
and the rates should all be the same. If an increase in rate is observed, fur-
ther checking of the assays is required.
The calculation of required enzyme levels allows economical use of
the auxiliary enzyme and will avoid many problems. McClure 3 has listed
the information that should be evaluated for a coupled assay and pre-
sented as part of the experimental methods. This includes (1) the units of
auxiliary enzyme added per milliliter of assay as described under the
experimental conditions; (2) the apparent Michaelis constants for the
measured products under the experimental conditions; (3) t* and the Fp
chosen on the basis of the calculations; and (4) the effect of the additional
auxiliary substrates on the activity of the primary enzyme. Most assays
currently used meet these criteria, having been determined by trial and
error. The treatment described here allows a more rational approach to
future design of coupled assays.
If a coupling enzyme is not available in sufficient quantity or some cir-
cumstance limits its use, the treatment of Kuchei et al. 7 will allow analysis
of a system where the activities of the primary and auxiliary enzymes are
similar. This technique would obviously not work with hysteretic en-
zymes but can be used with allosteric proteins as the primary enzyme. 7

Precautions
Once one is sure that sufficient auxiliary enzyme(s) is present to allow
accurate assays, there are a few other problems to be dealt with. The
40 INITIAL RATE METHODS [2]

reaction mixture for the primary e n z y m e should be preincubated with the

assay enzymes to determine whether any change occurs. Often the sub-
strates will be contaminated with a small amount of product, such as ADP
in ATP, which can react with the coupling system. The endogenous prod-
uct should be exhausted prior to actual assay. Often this results in actu-
ally better kinetic experiments, since no product will be present initially in
the reaction mixture. Also the system should be checked to see whether
there is a reaction observed in the absence of the assay enzymes. The
presence of other enzymes, particularly during purification, can give rise
to a blank rate that has to be considered and accounted for.
Another problem is contamination of the auxiliary enzymes by the pri-
mary enzyme. Often commercial enzymes will have a low contamination
of the activity being studied, but even a 0.1% contamination is often a
serious problem. This is more critical if the studies are being done with
auxiliary e n z y m e based on the calculations presented above. A related
problem is that the auxiliary enzymes will sometimes react with one o f the
primary substrates. Glucose-6-phosphate dehydrogenase has a small but
detectable reaction with glucose, s which limits the amount of e n z y m e that
can be used to couple enzymes such as hexokinase.
The final problem deals with interpretation of inhibition experiments
on the primary enzyme. If the inhibitor does not inhibit the auxiliary en-
zymes, no problems occur, since the activity of the primary e n z y m e will
be accurately measured. Often, however, an inhibitor that is a structural
analog o f a substrate will be an inhibitor of the auxiliary enzymes and will
cause an observed increase in the lag time. Generally, the advice has been
simply not to use coupled e n z y m e assays with inhibitors. The model
systems presented in the T h e o r y section do make useful predictions
regarding such effects. The inhibitor should be tested with the auxiliary
enzymes and the Ki and type o f inhibition be determined. If the inhibitor
is a competitive inhibitor of the auxiliary e n z y m e with respect to the mea-
sured product, both McClure 3 and Storer and Cornish-Bowden s suggest
that simply increasing the coupling enzyme will reduce the lag. In some
cases the lag may be too long to be able to reduce to a reasonable value,
but the addition of more auxiliary e n z y m e will usually allow the measure-
ments to be made. In general, similar considerations can be made for a
noncompetitive inhibitor. McClure's treatment suggests that an uncompet-
itive inhibitor will not alter the lag time since Kp and V2 change in a con-
stant ratio, but Storer and Cornish-Bowden 8 have shown that this is not
always true. It will be approximately true only if V2 is at least 10 times vl.
The effects of inhibitors on the auxiliary enzymes can be readily han-
dled using the techniques described here. The inhibitor can be determined
and compensated for by adding more auxiliary enzyme, and the lag time
[2] COUPLED ENZYME ASSAYS 41

can be analyzed on the actual assay so that the validity of the assay is as-
sured.

Summary
The amount o f auxiliary e n z y m e to be added to a assay system uti-
lizing a single coupled reaction can be calculated from
-2.303 log (1 - F,)Kp
V2 = (9)
t
based on McClure's 3 analysis where V2 is the number of units of E2, Fo is
the desired fraction o f the steady-state reaction of the primary e n z y m e to
be measured, Kp is the Michaelis constant for P for E2, and t is the desired
lag time. Alternatively the method of Storer and Cornish-Bowden 8 used
Eq. (17)
t* = 6Kp/vl (17)
where t* is the transient time, Kp is above, and Vl is the highest velocity of
the primary e n z y m e to be measured. A value for 6 is calculated at a speci-
fied time, and from Table I the ratio Vl/V2 is determined for a specified
v~/v~ ratio.
A plot of product (Q) appearance versus time allows evaluation of the
steady-state intermediate product level (Pss) and the lag time as a check on
the assumptions for the above equations. A check should always be done
to assure that the assays are linear with a reasonable lag time.
Storer and Cornish-Bowden's 8 treatment can be applied to systems
with several coupling enzymes, and the nomogram of McClure (Fig. 3) is
useful for a two-auxiliary e n z y m e system.
A check should always be done by adding a small amount of the pri-
mary e n z y m e product and determining that it reacts very rapidly with the
assay enzymes, ensuring that the assay system is actually working.

Concluding Remarks
The use of coupled e n z y m e assays affords a convenient, reliable
method of measuring the steady-state activity of an enzyme. Certain pre-
cautions must be taken to assure accuracy, and the system used should be
well documented as described above. Under proper conditions even inhi-
bition studies can be done with assurance of accurate measurements.
New assays can be designed with confidence avoiding trial-and-error
determinations.
42 INITIAL RATE METHODS [3]

Acknowledgments

This work was supported by Grants CA14030 awarded by the National Cancer Institute
and C-582 from the Robert A. Welch Foundation. B. W. B. and R. S. B. are Robert A.
Welch Foundation Predoctoral Fellows.

[3] S u m m a r y of Kinetic Reaction Mechanisms

By HERBERT J. FROMM

Kinetic mechanisms for enzyme-catalyzed reactions are ordinarily

proposed in order to explain initial-rate data. It was this very sort of at-
tempted correlation that led Brown 1 in 1902 to suggest that the enzyme
and its substrate must combine for a finite period before catalysis can
occur. Since then, a large number of kinetic mechanisms have been pro-
posed in order to explain the molecular mechanism of enzyme action.
An understanding of how the enzyme functions as a catalyst, and how
it is regulated in the cell requires in many cases a knowledge of the en-
zyme's kinetic mechanism. This information is necessary even in order to
evaluate inhibition constants for multisubstrate enzymes. Establishment
of the kinetic mechanism for an enzyme may provide information on,
among other things, the chemical mechanism, the nature of the transition
state, the geometry of the active site, substrate specificity, inhibition and
activation, acidic and basic groups associated with catalysis, allosteric
properties, and the mode of regulation.
Exactly why two different enzymes that catalyze single displacement
reactions should exhibit different pathways of enzyme and substrate in-
teraction leading to similar productive central complexes is a question
that remains to be answered. From the point of view of catalysis exclu-
sively, there seems to be no clear advantage for random substrate binding
to be preferred over ordered substrate binding by an enzyme, or vice
versa. Bell and Koshland 2 have summarized data for approximately 60
different enzyme systems in which there is strong evidence for the partici-
pation of covalent enzyme-substrate intermediates in catalysis. They
have also listed a number of ways in which such intermediates may be cat-
alytically important, but they also state that, considering the great cata-
lytic potential of enzymes, such intermediates are probably not essential
for catalysis. On the other hand, the order of substrate binding and prod-

t A. J. Brown, J. Chem. Soc. 81, 373 (1902).

2 R. M. Bell and D. E. K o s h l a n d , Jr., Science 172, 1253 (1971).

Copyright © 1979 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 63 All rights of reproduction in any form reserved.
ISBN 0-12-181963 9