Kinetic mechanism of 3-ketoacyl-(acyl-carrier-protein) reductase from Synechococcus sp. strain PCC 7942: A useful enzyme for the production of chiral alcohols

Article abstract of DOI:10.1016/j.molcatb.2010.12.013

Mathematical models and simulations have become indispensable tools for the characterization and optimization of enzymatic processes. Nonetheless, industrially relevant enzymes are often poorly characterized with respect to enzyme kinetics. For the description of bisubstrate reactions catalysed by oxidoreductases in many cases Michaelis-Menten kinetics is used, which is a significant simplification. The NADPH-dependent 3-ketoacyl-(acyl-carrier- protein) reductase (KR) from Synechococcus sp. strain PCC 7942 is an interesting biocatalyst for the asymmetric synthesis of a variety of chiral building blocks, such as ethyl (S)-4-chloro-3-hydroxybutanoate. Initial-rate analysis of the KR-catalysed reduction of ethyl 4-chloroacetoacetate to the corresponding (S)-alcohol gave families of straight lines in double-reciprocal plots consistent with a sequential mechanism being obeyed. Product inhibition studies revealed that the KR follows a steady-state ordered Bi Bi mechanism with NADPH binding first. This result was corroborated by fluorescence enhancement studies, which indicated that the cofactor can bind to the free enzyme. The dissociation constants for the binary NADPH-protein complex determined kinetically and by fluorescence titration were identical within experimental error (1.04 ± 0.35 mM and 1.01 ± 0.23 mM) and confirmed the accuracy of the obtained kinetic parameters.

Full text of DOI:10.1016/j.molcatb.2010.12.013

Journal of Molecular Catalysis B: Enzymatic 69 (2011) 89–94
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Kinetic mechanism of 3-ketoacyl-(acyl-carrier-protein) reductase from
Synechococcus sp. strain PCC 7942: A useful enzyme for the production of
chiral alcohols
∗
Kathrin Hölsch, Dirk Weuster-Botz
Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 September 2010
Received in revised form
Mathematical models and simulations have become indispensable tools for the characterization and
optimization of enzymatic processes. Nonetheless, industrially relevant enzymes are often poorly char-
acterized with respect to enzyme kinetics. For the description of bisubstrate reactions catalysed by
oxidoreductases in many cases Michaelis–Menten kinetics is used, which is a significant simplification.
The NADPH-dependent 3-ketoacyl-(acyl-carrier-protein) reductase (KR) from Synechococcus sp. strain
PCC 7942 is an interesting biocatalyst for the asymmetric synthesis of a variety of chiral building blocks,
such as ethyl (S)-4-chloro-3-hydroxybutanoate. Initial-rate analysis of the KR-catalysed reduction of ethyl
1
6 December 2010
Accepted 27 December 2010
Available online 7 January 2011
Keywords:
Chiral alcohols
4
-chloroacetoacetate to the corresponding (S)-alcohol gave families of straight lines in double-reciprocal
plots consistent with a sequential mechanism being obeyed. Product inhibition studies revealed that
the KR follows a steady-state ordered Bi Bi mechanism with NADPH binding first. This result was cor-
roborated by fluorescence enhancement studies, which indicated that the cofactor can bind to the free
enzyme. The dissociation constants for the binary NADPH–protein complex determined kinetically and
by fluorescence titration were identical within experimental error (1.04 ± 0.35 mM and 1.01 ± 0.23 mM)
and confirmed the accuracy of the obtained kinetic parameters.
Enzyme kinetics
Oxidoreductase
SDR superfamily
Steady-state ordered Bi Bi mechanism
©
2011 Published by Elsevier B.V.
1
. Introduction
Michaelis–Menten kinetics can also be applied to bisubstrate reac-
tions if pseudo-first-order reaction conditions are achieved and the
concentration of one of the substrates is kept at a constant, saturat-
ing surplus (≥10 × Km). However, this assumption is not valid for
the whole time course of preparative enzymatic reactions involving
oxidoreductases, because the nicotinamide cofactors are expensive
and are therefore not provided in excess quantities. Quite the con-
trary, cost-effective industrial bioreductions rely on efficient in situ
regeneration of catalytic amounts of reduced cofactors [6,7]. For
these reasons, the determination of the kinetic mechanism and the
subsequent development of a detailed mathematical model that
allows for the prediction of the enzymatic activity as function of
concentrations of substrates, cofactors and inhibiting compounds
are essential for process optimizations.
Mechanistic kinetic models have been shown to be valuable
tools for the optimization of various kinds of enzymatic reactions
e.g. [1–3]). Prerequisites for the accurate modelling of biocatalytic
(
processes are the detailed knowledge of the kinetic mechanism and
the determination of the kinetic parameters of all enzymes that are
involved.
Oxidoreductases are important biocatalysts for the industrial
production of chiral building blocks [4,5]. The asymmetric syn-
thesis of chiral alcohols catalysed by oxidoreductases is a classical
“
Bi Bi” reaction involving two substrates and two products: a keto
compound and a reduced cofactor, mostly NADH or NADPH, are
converted to the corresponding hydroxy compound and the oxi-
dized cofactor. The rate equations for bisubstrate mechanisms are
more complex than the Michaelis–Menten model, which is used
to describe monosubstrate reactions. In a simplifying approach,
Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-ECHB) is a key
intermediate in the synthesis of 3-hydroxy-3-methylglutaryl coen-
zyme A (HMG-CoA) reductase inhibitors, also known as statins
[
8]. This important chiral building block can be easily obtained
by asymmetric bioreduction of the prochiral ␤-keto ester ethyl
-chloroacetoacetate (ECAA). A kind of oxidoreductase that has
4
Abbreviations: ACP, acyl carrier protein; ECAA, ethyl 4-chloroacetoacetate;
ECHB, ethyl 4-chloro-3-hydroxybutyrate; KR, 3-ketoacyl-(acyl-carrier-protein)
reductase; SDR, short-chain dehydrogenase/reductase.
∗ Corresponding author. Tel.: +49 089 28915713; fax: +49 089 28915714.
E-mail address: D.Weuster-Botz@lrz.tum.de (D. Weuster-Botz).
recently attracted attention due to the ability to reduce ECAA to
the corresponding (S)-alcohol (Scheme 1A) is the fatty acid biosyn-
thetic enzyme 3-ketoacyl-[acyl-carrier-protein (ACP)] reductase
(KR, EC 1.1.1.100) [9–11]. This enzyme belongs to the dissociated
1
381-1177/$ – see front matter © 2011 Published by Elsevier B.V.
doi:10.1016/j.molcatb.2010.12.013

9
0
K. Hölsch, D. Weuster-Botz / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 89–94
Scheme 1. Reactions catalysed by 3-ketoacyl-[acyl-carrier-protein (ACP)] reductase (KR). (A) Reduction of ethyl 4-chloroacetoacetate. (B) Reduction of 3-ketoacyl-ACP. R
denotes the growing acyl chain.
type II fatty acid synthase (FAS II) system occurring in plants and
most bacteria [12] and is a member of the short-chain dehydro-
genase/reductase (SDR) superfamily. Its physiological function is
the reduction of 3-ketoacyl-ACP to (R)-3-hydroxyacyl-ACP with
NADPH as cofactor (Scheme 1B) [13,14].
assay mixtures contained 0–37.5 mM ECAA and 0–4 mM NADPH.
Controls lacking enzyme were routinely included. The samples
used for the determination of product inhibition patterns contained
+
0–16 mM NADP and 0–50 mM (S)-ECHB, respectively. All samples
were preincubated for 5 min before the reaction was initiated by the
addition of ␤-keto ester. Protein concentrations were determined
with the bicinchoninic acid (BCA) assay (Pierce, Rockford, USA) and
The enzymatic activities and enantiomeric excesses observed in
biocatalytic reductions of ECAA using KRs from different organisms
−1
−1
ranged between 0.1 and 250 U mg and 53.4 to >99.8% (S), respec-
tively [9,11]. Enantiomeric excesses higher than 99% were only
reported for enzymes originating from cyanobacteria [11], because
of which they are especially attractive for preparative applications.
Thus, more information about the kinetics of the enzymatic reac-
tion catalysed by cyanobacterial KRs would be valuable.
ranged from 10 to 50 mg L . Under these conditions, the enzyme
activity was proportional to the KR concentration. One unit (U) cor-
responds to the amount of enzyme converting 1 ␮mol substrate
◦
per min at 30 C. The oxidation of NADPH was measured photo-
metrically at 340 nm in microtiterplates. The reduction of ␤-keto
esters was measured by chiral gas chromatography. For this pur-
pose, enzyme reactions were carried out in 1.5 mL safe lock tubes
using a heating shaker (Eppendorf, Hamburg, Germany) at 600 rpm.
Since KRs are regarded as promising targets for the devel-
opment of antimicrobial agents [15], the kinetic mechanisms
of enzymes from several pathogenic microorganisms, such as
Streptococcus pneumoniae and Plasmodium falciparum, have been
determined [16–19]. The type of bisubstrate mechanism varied
between ordered Bi Bi and random Bi Bi reactions among KRs from
different species. This points to the fact that enzymes from different
sources have evolved to catalyse the same reaction through differ-
ent mechanisms. Here, we present the first determination of the
kinetic mechanism from a KR originating from the large phylum of
cyanobacteria. For this study, the KR from the freshwater cyanobac-
terium Synechococcus sp. strain PCC 7942 was chosen because it is
the best studied cyanobacterial KR so far [10,11].
◦
The reactions were stopped by adding ethyl acetate (−20 C) at a
volume ratio of 1:1 and the samples were immediately placed in an
ice chest. The extraction of substrates and products was performed
using a mixer mill (Retsch, Haan, Germany) for 10 min at 30 Hz.
2.4. Gas chromatographic analysis
A Varian CP-3800 gas chromatograph (GC) equipped with a
Flame Ionization Detector (FID) and a Lipodex-E (25 m × 0.25 mm
ID) column (Macharey-Nagel, Düren, Germany) was used for anal-
ysis of ␤-keto and ␤-hydroxy esters. The helium flow rate was
−
1
maintained at 4 mL min . Chiral analysis of ECAA, (R)- and (S)-
2
. Materials and methods
ECHB was performed as described by Bräutigam et al. [21].
2.1. Chemicals
2.5. Data analysis
Ethyl
purchased from Merck (Darmstadt, Germany). Ethyl (S)-
-chloro-3-hydroxybutyrate ((S)-ECHB) (∼96.0%) and ethyl
R)-4-chloro-3-hydroxybutyrate ((R)-ECHB) (98.0%) were obtained
from Sigma–Aldrich (Schnelldorf, Germany). The cofactors
NADP-Na (>95.0%) and NADPH-Na4 (>97.0%) were bought from
Calbiochem (part of Merck, Darmstadt, Germany) and Carl Roth
4-chloroacetoacetate
(ECAA)
(>98.0%)
was
Initial half saturation constants for ECAA and NADPH were
determined by varying the concentration of the substrate while
maintaining the cofactor NADPH at a fixed saturating level
4
(
(
≥10 × Km (app)) and vice versa [10]. Enzyme activities were
measured under initial reaction rate conditions in triplicates. Sub-
sequently, the data were fitted to the Michaelis–Menten equation
(
Eq. (1)):
(
Karlsruhe, Germany), respectively. All other chemicals were of
analytical grade from various suppliers.
Vmax[S]
Km (app) + [S]
v =
(1)
2.2. Cloning, expression and purification
where v is the reaction rate, Vmax is the maximum reaction rate, [S]
is the substrate concentration and Km (app) is the apparent half-
saturation constant.
To investigate further the kinetic mechanism and the order of
substrate binding, assays were performed where each substrate
The KR from Synechococcus sp. strain PCC 7942 was cloned, het-
erologously expressed in Escherichia coli (E. coli) and purified as
described previously [10]. The homogeneity of the protein prepa-
ration was analysed by 15% Tris–glycine SDS-PAGE stained with
silver according to the method by Heukeshofen and Dernik [20].
Since no contaminating protein was detected in the KR preparation,
the purity was judged to be near 100%.
was varied in turn with a fixed concentration of one of the prod-
ucts, either NADP+ or ␤-hydroxy ester. The second substrate was
kept constant at a sub-saturating concentration. The pattern of the
corresponding double-reciprocal plot was analysed and assigned
as linear competitive (Eq. (2)), uncompetitive (Eq. (3)), or non-
competitive/mixed (Eq. (4)) inhibition:
2.3. Enzyme assays
Vmax[S]
All assays for KR activity were performed in 0.1 M sodium phos-
phate buffer (pH 7.0) at 30 C in a reaction volume of 200 ␮L. The
v =
(2)
◦
Km(1 + ([I]/K )) + [S]
ic

K. Hölsch, D. Weuster-Botz / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 89–94
91
Vmax[S]
Km + [S](1 + ([I]/Kiu))
v =
v =
(3)
(4)
Vmax[S]
Km(1 + ([I]/K )) + [S](1 + ([I]/Kiu))
ic
where Kic is the inhibitor dissociation constant of the
enzyme–inhibitor complex and Kiu is the inhibitor dissocia-
tion constant of the enzyme–substrate–inhibitor complex. All
kinetic model parameters were identified by nonlinear regres-
sion analysis using the software program SigmaPlot 8.0 (SYSTAT
Software, Point Richmond, CA).
2.6. Determination of the NADPH dissociation constant
The dissociation constant of NADPH (K ) was determined by
d
fluorescence titration using a Tecan Infinite M200 reader (Tecan,
Männedorf, Switzerland). The fluorescence was excited at 280 nm
in the presence of increasing NADPH concentrations (0–1.04 mM)
in 0.1 M phosphate buffer (pH 7.0) at a protein concentration of
Fig. 1. Initial reaction rate pattern. Double-reciprocal plots of reaction rates with
ECAA as variable substrate at four concentrations of NADPH are shown. The fixed
NADPH concentrations were 0.25 mM (ꢀ), 0.5 mM (ꢁ), 2 mM (ꢀ), and 4 mM (᭹). The
inset shows replots of slopes (᭹) and intercepts (ꢀ) versus the reciprocal concen-
tration of the fixed substrate.
1
.29 ␮M. The dilution was kept below 5% and the fluorescence
emitted at 450 nm was measured. A control titration was carried
out in which the fluorescence of NADPH added in 1 ␮L-aliquots
to the buffer solution was determined. All data were corrected for
dilution, fluorescence of enzyme and buffer, and inner filter effects
of straight lines intersecting to the left of the ordinate (Fig. 1).
Replots of the slopes and intercepts against the reciprocal of the
fixed substrate concentration were also linear (Fig. 1 inset). When
the concentration of NADPH was varied and the ECAA concen-
tration was kept constant, the double reciprocal plots gave the
same pattern of lines intersecting in the second quadrant (data
not shown). These results are indicative of a sequential mecha-
nism in which both substrates are added to the enzyme before
a product is released and rule out the possibility of a ping-pong
mechanism, which yields parallel lines in a double-reciprocal plot.
Moreover, the results are incompatible with a rapid-equilibrium
ordered mechanism because an intersection point on the y-axis
would have been expected for one of the substrates [24].
[
22]:
(
Aex+Aem)/2
Fc = F · 10
(5)
where Fc is the corrected intensity, F the measured intensity, and
Aex and Aem are the absorbances of the sample at the excitation and
emission wavelength, respectively.
NADPH binding resulted in an enhanced fluorescence emission
at 450 nm due to energy transfer from tryptophan to NADPH. The
difference between the fluorescence measured in the presence of
the protein and the fluorescence of the control (ꢀFc) was calcu-
lated, which correlates with the amount of bound NADPH. The
dissociation constant Kd was determined by plotting the change in
emission against the free NADPH concentration. The SigmaPlot 8.0
software was used to fit the data to Eq. (6) by nonlinear regression:
3.2. Product inhibition studies
[
NADPHfree
]
ꢀ
Fc = ꢀFc max ·
(6)
To distinguish between different sequential bisubstrate mech-
Kd + [NADPHfree
Because a significant part of the added cofactor could have been
bound to the enzyme, the concentration of free NADPH was calcu-
lated as follows [23]:
]
anisms,
a product inhibition analysis under non-saturating
conditions was performed. The products of the reaction catalysed
by the KR were applied as inhibitors of the forward reaction (Fig. 2).
For the definition of non-saturating conditions, apparent half-
saturation constants were used (Km (app) NADPH: 0.40 ± 0.03 mM;
Km (app) ECAA: 8.3 ± 1.2 mM). Since the enantiomeric excess of (S)-
ECHB was determined to be 99.8 ± 0.4%, only the (S)-alcohol was
examined as inhibitor [10].
ꢀFc
[KRbound]
=
(7)
ꢀ
Fc max
[KRtotal
KRbound] = [NADPHbound
NADPHfree] = [NADPHtotal] − [NADPHbound
]
[
[
]
(8)
(9)
The double-reciprocal plots of the results showed that NADP+
was a linear competitive inhibitor with respect to NADPH (Fig. 2A),
but was a mixed inhibitor with respect to ECAA (Fig. 2C). In contrast,
]
At each cofactor concentration, only a negligible percentage
≤0.2%) of the total NADPH was enzyme-bound.
(
(
S)-ECHB was a mixed inhibitor of both NADPH (Fig. 2B) and ECAA
(
Fig. 2D). All replots of slopes and intercepts from product inhibition
3
. Results
studies were linear. The inhibition patterns and kinetic parameters
are summarized in Table 1.
Taken together, the initial reaction rate and product inhibition
patterns are consistent with a steady-state ordered Bi Bi mech-
anism with NADPH binding first, which is described in Eq. (10)
3.1. Initial reaction rate studies
As a first step in the determination of the kinetic mecha-
nism, the reaction rate with ECAA was measured at several fixed
[
24]:
V1V2([A][B] − ([P][Q])/Kg)
v =
(10)
KiAKmBV2 + KmBV2[A] + KmAV2[B] + (KmQV1[P])/Kg + (KmPV1[Q])/Kg + V2[A][B] + (KmQV1[A][P])/KiAKg + (KmAV2[B][Q])/KiQ + (V1[P][Q])/Kg + (V2[A][B][P])/KiP + (V1[B][P][Q])/KiiBKg
where V1 and V2 are the maximum reaction rates of the forward
and the reverse reaction, [A], [B], [P] and [Q] represent the con-
+
concentrations of NADPH. The double-reciprocal plots of ini-
tial reaction rate against ECAA concentration yielded a family
centrations of NADPH, ECAA, (S)-CHBE and NADP , KmA, KmB, KmP,
KmQ and KiA, KiB, K , K are the respective half-saturation and
iP
iQ

9
2
K. Hölsch, D. Weuster-Botz / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 89–94
Fig. 2. Product inhibition studies under non-saturating conditions. The kinetic data are displayed as double reciprocal plots of the reaction rate versus substrate concentrations
at different concentrations of the product inhibitor. For each plot, one substrate was kept at a constant non-saturating level while the second substrate and the product
inhibitor concentrations were varied. (A) NADP+ is a competitive inhibitor to NADPH. ECAA was kept constant at 4 mM while varying NADPH and NADP concentrations
+
(
(
ꢀ
ꢀ
᭹ = 0, ꢀ = 2, ꢁ = 4, ꢀ = 8, ꢂ = 16 mM). (B) (S)-ECHB is a mixed-type inhibitor to NADPH. ECAA was kept constant at 4 mM while varying NADPH and (S)-ECHB concentrations
+
+
᭹ = 0, ꢀ = 12.5, ꢁ = 25, ꢀ = 50 mM). (C) NADP is a mixed-type inhibitor to ECAA. NADPH was kept constant at 0.4 mM while varying ECAA and NADP concentrations (᭹ = 0,
= 2, ꢁ = 4, ꢀ = 8, ꢂ = 16 mM). (D) (S)-ECHB is a mixed-type inhibitor to ECAA. NADPH was kept constant at 0.5 mM while varying ECAA and (S)-ECHB concentrations (᭹ = 0,
= 12.5, ꢁ = 25, ꢀ = 50 mM). The insets show replots of slopes (᭹) and intercepts (ꢀ) versus inhibitor concentrations.
dissociation constants, and Kg is the equilibrium constant of
the reaction. Under initial reaction rate conditions, this equation
reduces to
model parameters. The maximum reaction rate of the reverse reac-
−
1
tion was estimated to be 0.15 U mg , which equals about 0.4%
of the maximum reaction rate of the forward reaction. Because
both products of the reverse reaction, ECAA and NADPH, have
only limited stability under the assay conditions [25,26], long
incubation times were incompatible with accurate results. There-
fore, kinetic studies of the reverse reaction were not pursued
further.
V1[A][B]
v = K
(11)
iAKmB + KmB[A] + KmA[B] + [A][B]
for the forward reaction and
V2[P][Q]
v = K
(12)
The kinetic data obtained for the forward reaction in the absence
of products were globally fitted to the equation for the steady-state
ordered Bi Bi mechanism (Eq. (11)) (Fig. 3). The derived kinetic con-
iAKmB + KmQ[P] + KmP[Q] + [P][Q]
for the reverse reaction.
As the very low rate of the reverse reaction precluded detailed
kinetic analyses, it was not possible to identify all of the kinetic
−
1
stants are: V = 36.79 ± 2.11 U mg , KmA (NADPH) = 0.33 ± 0.9 mM,
1
KmB (ECAA) = 6.94 ± 1.34 mM, and K = 1.04 ± 0.35 mM.
iA
Table 1
Product inhibition patterns and derived inhibition constants.
Varied substrate
Product (inhibitor)
Inhibition pattern
Inhibition constant
Kic (mM)
Kiu (mM)
NADPH
NADPH
ECAA
NADP+
Competitive
6 ± 1
47 ± 20
8 ± 3
–
210 ± 98
68 ± 59
(S)-ECHB
Mixed (non-competitive)
Mixed (non-competitive)
Mixed (non-competitive)
NADP+
ECAA
(S)-ECHB
94 ± 54
115 ± 71

K. Hölsch, D. Weuster-Botz / Journal of Molecular Catalysis B: Enzymatic 69 (2011) 89–94
93
consistencies of these independently determined parameters and
of the dissociation constants of the binary NADPH–protein complex
determined via kinetic experiments (1.04 ± 0.35 mM) and fluores-
cence enhancement (1.01 ± 0.23 mM) verify the correctness of the
obtained kinetic model.
The steady-state ordered Bi Bi mechanism is common among
nicotinamide nucleotide-dependent oxidoreductases and has also
been proposed for the KR from the malaria parasite P. falciparum
[
19], for the related enoyl-ACP reductase from Brassica napus (rape)
[
27], and for other members of the SDR superfamily (e.g. [28–30]).
Structural studies on the KR from E. coli provided evidence for
considerable conformational changes after binding of the cofac-
tor leading to the hypothesis of an ordered kinetic mechanism in
which the binding of NADPH is an essential prerequisite for the
binding of the keto substrate [16]. In contrast, rapid-equilibrium
and steady-state random Bi Bi mechanisms were proposed for
the KRs from S. pneumoniae [17] and Mycobacterium tuberculo-
sis [18], respectively. The observed product inhibition patterns in
this study eliminated the possibility of both random mechanisms.
In the rapid-equilibrium random mechanism (without formation
of dead-end complexes) all product inhibition patterns would be
competitive and the steady-state random mechanism would result
in non-competitive inhibition in case of all four substrate–product
combinations [31].
From an application-oriented point of view, the presented
results revealed that both reaction products caused only a weak
inhibition of the cyanobacterial KR. High apparent inhibition con-
stants in the range of 7–200 times the Km of the corresponding
substrate were observed. The enzyme showed a steep activity
decrease at substrate and cofactor concentrations below 10 mM
ECAA and/or 1 mM NADPH, respectively. To realize an economi-
cally feasible process involving the KR, a high NADPH concentration
and an efficient in situ regeneration of the oxidized cofactors would
be necessary. The application of an artificial fusion protein between
the KR from Synechococcus sp. strain PCC 7942 and a cofactor regen-
erating enzyme has been shown to result in higher reaction rates
than an equimolar mixture of the independent enzymes [32]. The
identification of the reaction mechanism of the KR is a first step
toward a deeper understanding of this finding and the modelling
of the coupled enzymatic reaction.
Fig. 3. Graphical representation of the initial reaction rate data obtained in the
absence of products. The experimental data are shown as dots and the lines repre-
sent the predicted values obtained by fitting the data points to Eq. (11) by nonlinear
regression analysis.
3.3. NADPH binding studies
Interaction between the free enzyme and NADPH was further
demonstrated by the transfer of energy between the protein and
the cofactor. Excitation of tryptophan fluorescence at 280 nm in the
presence of increasing NADPH concentrations caused the cofactor
to emit an increasing amount of light at 450 nm and gave evidence
of the formation of a binary enzyme-cofactor complex (Fig. 4).
Because NADPH concentrations higher than 1.04 mM exceeded
the absorbance measurement range of the photometer, the flu-
orescence values measured at these concentrations could not be
corrected for inner filter effects and were not used for the deter-
mination of the dissociation constant Kd of the enzyme-NADPH
complex. Nonlinear regression analysis of the data yielded a dis-
sociation constant Kd of 1.01 ± 0.23 mM. This result is in good
agreement with the parameter KiA (1.04 ± 0.35 mM) of the ordered
Bi Bi mechanism, which represents the dissociation constant of the
first substrate to bind to the free enzyme.
4
. Discussion
The results from the initial reaction rate, product inhibition,
5. Conclusion
and NADPH-binding studies led to the conclusion that the KR from
the freshwater cyanobacterium Synechococcus sp. strain PCC 7942
operates by a steady-state ordered Bi Bi mechanism involving a
ternary complex with NADPH binding before the keto substrate.
The estimated maximum reaction rate of the forward reaction
In summary, the KR from the freshwater cyanobacterium
Synechococcus sp. strain PCC 7942 has been shown to obey a steady-
state ordered Bi Bi mechanism. The correct assignment of the
kinetic mechanism is the fundamental basis for the mathematical
description and optimization of large-scale KR-catalysed reduction
reactions.
−1
(
36.79 ± 2.11 U mg ) is not significantly different from the specific
activity of the KR that was determined in previous experiments
with ECAA and NADPH in excess (38.29 ± 2.15 U mg−1) [10]. The
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(
Fig. 4. Fluorescence titration with NADPH. The dots represent the measured change
in emission (ꢀFc) and the line represents the predicted values obtained by fitting
the experimental data to Eq. (6) by nonlinear regression analysis.

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4
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