Download Effect of non-ionic detergents on apparent enzyme mechanism

Protein Engineering vol.11 no.8 pp.609–611, 1998
SHORT COMMUNICATION
Effect of non-ionic detergents on apparent enzyme mechanism:
V121A mutant of Streptomyces cholesterol oxidase endowed with
enhanced sensitivity towards detergents
Yoshiaki Nishiya1, Mitsuo Yamashita2,
Yoshikatsu Murooka2, Isao Fujii3 and Noriaki Hirayama3
Tsuruga Institute of Biotechnology, Toyobo Co., Ltd, Toyo-cho, Tsuruga,
Fukui 914, 2Department of Biotechnology, Graduate School of Engineering,
Osaka University, Yamadaoka, Suita, Osaka 565 and 3Department of
Biological Science and Technology, Tokai University, Nishino, Numazu,
Shizuoka 410-03, Japan
1To
whom correspondence should be addressed
One of the mutants of Streptomyces cholesterol oxidase with
the Val121Ala mutation (V121A) was kinetically analysed.
Although the reaction rate–substrate concentration curve
of wild type follows a simple Michaelis–Menten equation,
that of V121A is sigmoidal. The cooperativity was apparent
and caused by non-ionic detergents that were used as a
solvent of cholesterol. The concentration dependence of
V121A on detergents was more significant than that of
wild type, although the reaction rates of both enzymes
decrease as the concentrations of detergents increase.
Further experiments suggested that less hydrophobic
interactions between V121A and detergents should be
responsible for the apparent cooperativity. Since Val121 is
in a hydrophobic loop located near the active site, the
mutational effect is structurally discussed.
Keywords: cholesterol oxidase/Streptomyces/mutational effect/
apparent cooperativity/non-ionic detergent
Introduction
Cholesterol oxidase (EC 1.1.3.6; cholesterol:oxygen oxidoreductase) catalyzes the oxidation of cholesterol (5-cholestan-3ol) and forms equimolar amounts of cholest-4-en–3-one and
hydrogen peroxide (Stadtman et al., 1954; Uwajima et al.,
1973, 1974). This enzyme is useful for the clinical determination of serum cholesterol by coupling with related enzymes
(Allain et al., 1974). Both of the Streptomyces and Brevibacterium enzymes are commercially produced and applied as
diagnostic reagents. Brevibacterium cholesterol oxidase
(ChoB) has been studied by X-ray crystallography, which
provided a complete structural description of the enzyme
(Vrielink et al., 1991). Substrate binding features and the
catalytic mechanism have also been discussed (Li et al., 1993).
Recently, we have succeeded in the improvement of thermal
stability of the Streptomyces enzyme (ChoA) by means of in
vitro mutagenesis techniques (Nishiya et al., 1997). Four
thermostable mutants were obtained and one amino acid
residue was replaced in each mutant. All of the amino
acid substitutions were located within a limited region (from
residues 103 to 145) of 546 residues. A three-dimensional
structure of ChoA was constructed on the basis of the X-ray
analysis of a complex between ChoB and dehydroisoandrosterone, a competitive inhibitor of cholesterol oxidase, and the
mutational effects were structurally interpreted.
In this report, one of the thermostable ChoA mutants with
the Val121Ala mutation, designated V121A, is kinetically
© Oxford University Press
analysed. Val121 is located in an external loop from residues
116 to 127 (Figure 1). This loop corresponds to the entrance
to the active site of cholesterol oxidase and is relatively
flexible, judging from high temperature factors from the Xray analysis.
Materials and methods
The purified wild-type and V121A ChoAs were prepared as
previously described (Nishiya et al., 1997).
Cholesterol oxidase activities and reaction rates of ChoAs
were measured by the method of Allain et al. (1974) as
previously described (Nishiya et al., 1997). Cholesterol was
dissolved in an aqueous solution of non-ionic detergents or in
isopropyl alcohol and used as the aqueous substrate. Enzyme
solutions were prepared by dilution with 20 mM potassium
phosphate (pH 7.0) containing 0.2% bovine serum albumin.
The reaction mixtures finally contained 50 mM potassium
phosphate (pH 7.0), varying concentrations of cholesterol
dissolved in an aqueous solution of non-ionic detergents or in
isopropyl alcohol, 0.45 mM 4-aminoantipyrine, 2.0 mM phenol
and 5 U/ml horseradish peroxidase. The appearance of quinoneimine dye formed by coupling with 4-aminoantipyrine, phenol
and peroxidase was measured at 500 nm and 37°C by spectrophotometry.
Reagents used were purchased from Nacalai Tesque Co.,
Ltd, Kyoto, Japan.
Results and discussion
Thermostable mutants obtained by protein engineering often
show significant changes in other physicochemical properties.
No such additional change was observed for the V121A
enzyme. The specific activities (with 1 mM cholesterol dissolved in an aqueous solution of Triton X-100 as the substrate,
at 37°C and pH 7.0) of wild type and V121A were 67.7 and
72.0 U/mg, respectively. The effects of pH on the activity and
stability, and the optimum temperature of V121A were also
the same level as those of the wild type (Nishiya et al., 1997).
When analysed in SDS and native polyacrylamide gels, the
wild-type and mutant proteins were indistinguishable in their
electrophoretic properties (data not shown). From these, we
concluded that no extensive conformational changes of the
cholesterol oxidase structure were induced by the amino acid
substitution.
Km value of V121A for cholesterol could not be exactly
determined as described in our previous paper (Nishiya et al.,
1997). In order to understand the kinetic behaviour of the
V121A mutant, the reaction rate–substrate concentration curve
was compared with that of wild type (Figure 2). Cholesterol
was dissolved in an aqueous solution of non-ionic detergent
Triton X-100 and used as the aqueous substrate. The concentration of Triton X-100 in the experiment is constant and the
ratio of the concentrations of the detergent to that of cholesterol
is high at a low concentration of cholesterol. Although the curve
of wild type follows a simple Michaelis–Menten equation, that
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Y.Nishiya et al.
Fig. 1. ChoA structure derived from homology modelling. Substrate
molecule (dehydroisoandrosterone) is represented by a space-filling model.
The side chain of the V121 residue is shown by stick drawing.
Fig. 2. Reaction rate–substrate concentration curves of wild type and
V121A. Triton X-100 was used as a solvent of cholesterol. Experiments
were performed as described in the Materials and methods section, and
0.05 U/ml of each enzyme was used for measuring the reaction rates. A
final concentration of Triton X-100 was 0.077% and calculated to be
1.1 mM using averages of molecular weights. d; wild type, s; V121A.
of V121A is sigmoidal. The reaction rates of V121A at low
concentrations of cholesterol are very slow. Since ChoA is a
monomer enzyme, the unexpected sigmoidal curve cannot be
due to cooperativity and this unusual phenomenon must have
an alternative explanation.
The substrate-binding curves for wild type and V121A were
measured with dehydroisoandrosterone and they are essentially
the same as those with cholesterol (data not shown). Other
non-ionic detergents with different chemical structures, such
as Triton N-101, Emulgen 950 and Tween 85, also cause
similar cooperativity of V121A (data not shown). These
indicate that the kinetic behaviour of V121A is not dependent
on kinds of substrates and non-ionic detergents.
The effect of detergent concentration on kinetics were
investigated using the assay reagents with 0.02 mM cholesterol
and varying concentrations of Triton X-100. Time courses of
the absorbance changes are shown in Figure 3. At the low
concentration of cholesterol V121A exhibits much lower
reaction rates than wild type, and the reaction rates of both
enzymes decrease as the concentration of Triton X-100
610
Fig. 3. Effects of concentration of detergent on the reaction rates. Triton
X-100, X-165 or X-305 was used as the solvent for cholesterol.
Experimental conditions were described as Figure 2. The concentrations of
detergents were calculated using averages of molecular weights. d; wild
type, s; V121A.
increases. The concentration dependence of V121A is more
significant than that of wild type.
Triton X-100 consists of a hydrophobic p-octylphenyl group
and a polyethylene glycol chain. In Triton X-100 the average
number of ethylene glycol units in the chain (n) is 10. To
investigate the effect of the length of the chain moiety, the
reaction rates with Triton X-165 (n 5 16), and Triton X-305
(n 5 30) are compared. Figure 3 shows that Tritons X-100,
X-165 and X-305 effect the reaction rates in a similar manner
and the magnitude of the effect is dependent on the concentration. As the suppression of the reaction, however, depends on
the length of the chain, the effect of the detergents may be
mainly a hydrophobic one due to the chain moiety.
Small amounts of cholesterol can be dissolved in aqueous
alcohol solution as well as aqueous solution of non-ionic
detergents. To compare the effects of alcohols and non-ionic
detergents on the reaction rate, isopropyl alcohol was used
as the solvent for cholesterol. The reaction rate–substrate
concentration curves for wild type and V121A are shown in
Figure 4. Although the rate with alcohol is much higher than
that with the non-ionic detergent, the curve of V121A is not
Effect of non-ionic detergents on apparent enzyme mechanism
Hydrophobicity of the hydrophobic core is reduced by the
substitution with alanine at position 121, and the reduced
hydrophobicity is responsible for the abnormal enzyme kinetics, since it would be more difficult for the mutant enzyme to
take up the substrate from micelles. It is noteworthy that the
substitution from Val to Ala in the loop region on the surface
of enzyme seems to be marginal but the effect on detergents
is profound. The present results warn that the reaction kinetics
for a system with detergents must be treated with much care
especially at low concentrations of substrate.
References
Fig. 4. Reaction rate–substrate concentration curves of wild type and
V121A. Isopropyl alcohol was used as the solvent for cholesterol.
Experimental conditions were described as Figure 2. A final concentration
of isopropyl alcohol was 1.5% and calculated to be 0.20 M. d; wild type,
s; V121A.
sigmoidal in this case and essentially the same with that of
wild type. This suggests that the cooperative phenomenon of
V121A is dependent on non-ionic detergents.
V121 is in a hydrophobic loop which is located near the
active site. The loop covers the active site as shown in Figure 1.
As the side chain of V121 is exposed on the surface of the
enzyme, substitution of Ala for Val in V121A does not affect
the structure of the binding site in the enzyme and the enzyme
activity is almost the same as that of wild type. The protruding
side chain of V121, however, would be more favorable for
hydrophobic interactions with detergents.
Without non-ionic detergents or higher alcohols cholesterol
cannot be dissolved in solution and the enzyme reaction will
not proceed. Aqueous solution non-ionic detergents and higher
alcohols form micelles in which hydrophobic cholesterol can
be included. Enzyme must deprive cholesterol from the micelle
to undergo enzyme reaction and the affinity of enzyme for
detergents should be crucial for the reaction. Since valine is
protruding on the surface of enzyme, it is favorable for
hydrophobic interactions with detergents and to deprive cholesterol from the micelle. Alanine is, however, less favorable for
these interactions.
In the solution with more hydrophobic detergents, V121A
cannot efficiently uptake cholesterol. On the other hand, V121A
can uptake cholesterol in the solution with less hydrophobic
detergents such as isopropyl alcohol. The interaction between
V121A and cholesterol is also highly dependent on the
concentration of detergents. Although there is less chance for
V121A to interact with cholesterol under a high concentration
of detergents, under a low concentration of detergents V121A
can uptake cholesterol from the medium and the enzymatic
reaction will proceed with the rate almost identical with that
of V121.
The present study has suggested that less hydrophobic
interactions between V121A and non-ionic detergents should
be responsible for the apparent sigmoidal behaviour of V121A.
The residue V121 is located in a loop, and the side chain is
involved in a hydrophobic core near the active site. This
hydrophobic core may play an important role in extracting the
substrate from micelles in which the substrate is surrounded
by the detergent molecules used in the reaction mixture.
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Received February 18, 1998; revised April 2, 1998; accepted April 3, 1998
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