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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 609 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. Allain,C.C., Poon,L.S., Chan,C.G.S., Richmond,W. and Fu,P.C. (1974) Clin. Chem., 20, 470–475. Li,J., Vrielink,A., Brick,A. and Blow,D.M. (1993) Biochemistry, 32, 11507– 11515. Nishiya,Y., Harada,N., Teshima,S., Yamashita,M., Fujii,I., Hirayama,N. and Murooka,Y. (1997) Protein Engng, 10, 231–235. Stadtman,T.C., Cherkes,A. and Anfinsen,C.B. (1954) J. Biol. Chem., 206, 511–512. Uwajima,T., Yagi,H., Nakamura,S. and Terada,O. (1973) Agr. Biol. Chem., 37, 2345–2350. Uwajima,T., Yagi,H. and Terada,O. (1974) Agr. Biol. Chem., 38, 1149–1156. Vrielink,A., Lloyd,L.F. and Blow,D.M. (1991) J. Mol. Biol., 219, 533–554. Received February 18, 1998; revised April 2, 1998; accepted April 3, 1998 611