嗜热蛋白酶PhpI分子克隆及突变体Y120P对酶学性质影响
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摘要
蛋白酶是一类重要的水解酶,它能催化蛋白质降解为多肽段和氨基酸,具有重要的生理功能,并在工业上有广泛的用途。基于其分布广、种类多的特点,发现新型蛋白酶,确定其分子作用机制及生物学意义成为重要的研究课题。超嗜热古菌Pyrococcus horikoshii中的嗜热蛋白酶PhpI是一类新型的蛋白酶,它属于DJ-1/ThiJ/PfpI超家族。为了解该酶结构和功能的关系以及催化机制,我们应用分子生物学技术及蛋白质工程等方法对其进行了较为系统的酶学性质研究。
我们将超嗜热蛋白酶PhpI的基因在常温菌E.coli中进行克隆表达,证明重组酶主要以十二聚体的形式存在。酶学性质分析发现:该蛋白酶的最适温度为80℃,最适pH为8.0,在pH 7.5-8.5范围内相对活力保持在80%以上;该蛋白酶对多种有机溶剂都具有较高的抗性;0.2%-5.0%的Tween-20和Triton X-100对酶有激活作用;该酶的底物作用谱宽,不仅对天然蛋白酶底物有水解活力,同时还具有氨基肽酶和内切酶的水解活力;稳态动力学分析表明其最适氨基肽酶底物为L-R-AMC,kcat/Km:0.052μM-1min-1;其最适内切酶底物为L-AAFR-AMC,kcat/Km:0.011μM-1min-1。
晶体结构分析表明,天然酶的120位点处于底物结合口袋的入口处并与催化基团Cys100形成主链氢键,推测可能对酶催化有重要的影响。我们构建的突变体Y120P,既消除了天然酶在底物结合口袋入口处Tyr120侧链苯环的空间位阻,也破坏了主链氢键的形成。研究该位点对酶催化及底物选择性的影响时发现,Y120P在保持了对天然大分子底物的水解活力基础上,对短肽底物催化能力也有提高。动力学数据表明其催化L-R-AMC的kcat值约是野生型的7倍,Km基本没有变化;催化L-AAFR-AMC的kcat约是野生型的10倍,Km约为野生型的1/2。结合生物信息学分析发现:120位点突变为P后底物结合口袋的直径增加了3.7 ?,且打断了与Cys100的主链氢键作用,更有利于较大底物分子的结合和催化作用。本研究对深入了解这类新型半胱氨酸蛋白酶催化机制奠定了基础。
Proteases are a group of proteins that catalyze the cleavage of peptide bonds in peptides, polypeptides, and proteins using a hydrolysis reaction. From a biological standpoint, these enzymes are critical for cellular survival, particularly in removal of denatured proteins during stress events or of proteins that have completed their functions. Various proteases play distinct roles in the degradation of proteins, including protease that break down proteins and peptidases that break down the resulting oligopeptide products to single residues. So it is an important research direction of such enzymes. The cysteine protease PhpI (Pyrococcus horikoshii protease I) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 belongs to the PfpI family of DJ-1/ThiJ/PfpI superfamily, which is famous for the similarity of the 3D structures between members but with various functions. In order to understanding the structure and function of enzymes and the relationship of the catalytic mechanism, we utilized the moleculer technology and the method of protein engineering to study the biochemical characterization.
The thermophilic protease gene PhpI has been successfully cloned and over expressed in soluble form. After the heat treatment and gel exclusion chromatography, the purified enzyme was in the form of dodecamer, which had been identified by Western Blotting and enzyme overlay analysis. Biochemical characterization revealed that the optimal reaction temperature and pH values were 80 oC and 8.0 respectively. It maintained relative activity above 80% in the range of pH 7.5-8.5. The protease PhpI was obviously inhibited by the cysteine protease inhibitors (e.g. DTT, Cys-HCl, 2-Me and IAA). It was likely that these inhibitors covalently modified the -SH of Cys100 and prevented the catalysis. The enzyme was strongly activated by 0.2%-5.0% Tween-20 and Triton X-100, while with high ability of organic reagent resistance. PhpI was highly inhibited by divalent metal ions, such as Zn2+, Cu2+, Fe3+, Ni2+, Co2+. In this study, we showed that PhpI was not only an aminopeptidase with broad specificity but also an endopeptidase preferred the arginine acid on the P1 site. The protease hydrolyzed the optimum synthetic substrate L-R-AMC with a kcat of 0.6348 min-1, a Km of 12μM and a kcat/Km of 0.052μM-1min-1 as an aminopeptidase. And the kinetic parameters of the best Endopeptidase substrate L-AAFR-AMC were as followed: kcat = 0.112 min-1, Km = 10μM, kcat/Km = 0.011μM-1min-1.
Based on the 3D structure of PhpI, it was found that the large phenyl of Tyr 120 was at the entrance of the pocket, as a result that the diameter of the tunnel-like substrate binding pocket was about 8.2 ?. Meanwhile, there was a hydrogen bond between the carbonyl oxygen of Cys 100 and the nitrogen of Tyr 120. To investigate the function of Tyr 120 in the catalysis of PhpI, we designed and constructed the mutant Y120P in order to eliminate the side chain obstacle of Tyr 120 and to interrupt the hydrogen bond on the main chain.
Compared to the parent enzyme, the properties of mutant Y120P had distinctly changed. Biochemical characterization showed that the best reaction temperature was 75 oC, about 5 oC lower than that of the PhpI, and it could keep higher relative activity between 30-70 oC. The results indicated that the substitution at the position might affect the thermo stability of the enzyme, resulting in the high hydrolysis activity in a larger range of temperature. Its optimum pH value was 7.5, which was 0.5 lower than parent enzyme and keeping relative activity above 80% in the range of pH 6.0 to 8.0; the pK1 of mutant Y120P decreased 1 point, which implied that the amino polarity at the position 120 might cause the changes of electrostatic environment at the activity center. The assay of the substrates specificity identified that the Y120P maintained the hydrolysis ability against the natural substrate gelatin, and increased the relative activity on the shorter peptide substrates. The kinetic results showed that the kcat cleaving L-R-AMC was 7-fold higher than that of wild type, which was 4.37 min-1, while holding similar Km. On the other hand, a 10-fold increase in kcat was observed on the hydrolysis of L-AAFR-AMC, it was also worthy noting that the Km value was only 1/2 compare with that of Wild type PhpI. The results above revealed that the major increase in the activities were due to the higher kcat values, indicating the Tyr->Pro mutagenesis mainly accellrated the catalysis of the enzyme but not substrate binding process. However, a 1/2 decrease in Km for L-AAFR-AMC also showed that this mutant was more favourable for bigger substrate binding. Molecular docking results also confirmed our deduction. Interupting the main chain hydrogen bond and eliminating the side chain benzyl of Tyr120 (Tyr->Pro) made it easier for substrate approach active site Cys100 and oxyanion hole Gly70, thereby increasing the enzyme catalysis. In the meanwhile, the mutant enlarged the binding entrance, thereby accelerating the substrate binding.
In conclusion, we have successfully obtained the recombined thermophilic protease PhpI. Baced on the biochemical characterization and the analysis of the 3D structure, we identified the importance of the position120 which had a close relationship with the catalysis and the substrate specificity. Our study should be useful for the rational design of new enzymes.
我们将超嗜热蛋白酶PhpI的基因在常温菌E.coli中进行克隆表达,证明重组酶主要以十二聚体的形式存在。酶学性质分析发现:该蛋白酶的最适温度为80℃,最适pH为8.0,在pH 7.5-8.5范围内相对活力保持在80%以上;该蛋白酶对多种有机溶剂都具有较高的抗性;0.2%-5.0%的Tween-20和Triton X-100对酶有激活作用;该酶的底物作用谱宽,不仅对天然蛋白酶底物有水解活力,同时还具有氨基肽酶和内切酶的水解活力;稳态动力学分析表明其最适氨基肽酶底物为L-R-AMC,kcat/Km:0.052μM-1min-1;其最适内切酶底物为L-AAFR-AMC,kcat/Km:0.011μM-1min-1。
晶体结构分析表明,天然酶的120位点处于底物结合口袋的入口处并与催化基团Cys100形成主链氢键,推测可能对酶催化有重要的影响。我们构建的突变体Y120P,既消除了天然酶在底物结合口袋入口处Tyr120侧链苯环的空间位阻,也破坏了主链氢键的形成。研究该位点对酶催化及底物选择性的影响时发现,Y120P在保持了对天然大分子底物的水解活力基础上,对短肽底物催化能力也有提高。动力学数据表明其催化L-R-AMC的kcat值约是野生型的7倍,Km基本没有变化;催化L-AAFR-AMC的kcat约是野生型的10倍,Km约为野生型的1/2。结合生物信息学分析发现:120位点突变为P后底物结合口袋的直径增加了3.7 ?,且打断了与Cys100的主链氢键作用,更有利于较大底物分子的结合和催化作用。本研究对深入了解这类新型半胱氨酸蛋白酶催化机制奠定了基础。
Proteases are a group of proteins that catalyze the cleavage of peptide bonds in peptides, polypeptides, and proteins using a hydrolysis reaction. From a biological standpoint, these enzymes are critical for cellular survival, particularly in removal of denatured proteins during stress events or of proteins that have completed their functions. Various proteases play distinct roles in the degradation of proteins, including protease that break down proteins and peptidases that break down the resulting oligopeptide products to single residues. So it is an important research direction of such enzymes. The cysteine protease PhpI (Pyrococcus horikoshii protease I) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 belongs to the PfpI family of DJ-1/ThiJ/PfpI superfamily, which is famous for the similarity of the 3D structures between members but with various functions. In order to understanding the structure and function of enzymes and the relationship of the catalytic mechanism, we utilized the moleculer technology and the method of protein engineering to study the biochemical characterization.
The thermophilic protease gene PhpI has been successfully cloned and over expressed in soluble form. After the heat treatment and gel exclusion chromatography, the purified enzyme was in the form of dodecamer, which had been identified by Western Blotting and enzyme overlay analysis. Biochemical characterization revealed that the optimal reaction temperature and pH values were 80 oC and 8.0 respectively. It maintained relative activity above 80% in the range of pH 7.5-8.5. The protease PhpI was obviously inhibited by the cysteine protease inhibitors (e.g. DTT, Cys-HCl, 2-Me and IAA). It was likely that these inhibitors covalently modified the -SH of Cys100 and prevented the catalysis. The enzyme was strongly activated by 0.2%-5.0% Tween-20 and Triton X-100, while with high ability of organic reagent resistance. PhpI was highly inhibited by divalent metal ions, such as Zn2+, Cu2+, Fe3+, Ni2+, Co2+. In this study, we showed that PhpI was not only an aminopeptidase with broad specificity but also an endopeptidase preferred the arginine acid on the P1 site. The protease hydrolyzed the optimum synthetic substrate L-R-AMC with a kcat of 0.6348 min-1, a Km of 12μM and a kcat/Km of 0.052μM-1min-1 as an aminopeptidase. And the kinetic parameters of the best Endopeptidase substrate L-AAFR-AMC were as followed: kcat = 0.112 min-1, Km = 10μM, kcat/Km = 0.011μM-1min-1.
Based on the 3D structure of PhpI, it was found that the large phenyl of Tyr 120 was at the entrance of the pocket, as a result that the diameter of the tunnel-like substrate binding pocket was about 8.2 ?. Meanwhile, there was a hydrogen bond between the carbonyl oxygen of Cys 100 and the nitrogen of Tyr 120. To investigate the function of Tyr 120 in the catalysis of PhpI, we designed and constructed the mutant Y120P in order to eliminate the side chain obstacle of Tyr 120 and to interrupt the hydrogen bond on the main chain.
Compared to the parent enzyme, the properties of mutant Y120P had distinctly changed. Biochemical characterization showed that the best reaction temperature was 75 oC, about 5 oC lower than that of the PhpI, and it could keep higher relative activity between 30-70 oC. The results indicated that the substitution at the position might affect the thermo stability of the enzyme, resulting in the high hydrolysis activity in a larger range of temperature. Its optimum pH value was 7.5, which was 0.5 lower than parent enzyme and keeping relative activity above 80% in the range of pH 6.0 to 8.0; the pK1 of mutant Y120P decreased 1 point, which implied that the amino polarity at the position 120 might cause the changes of electrostatic environment at the activity center. The assay of the substrates specificity identified that the Y120P maintained the hydrolysis ability against the natural substrate gelatin, and increased the relative activity on the shorter peptide substrates. The kinetic results showed that the kcat cleaving L-R-AMC was 7-fold higher than that of wild type, which was 4.37 min-1, while holding similar Km. On the other hand, a 10-fold increase in kcat was observed on the hydrolysis of L-AAFR-AMC, it was also worthy noting that the Km value was only 1/2 compare with that of Wild type PhpI. The results above revealed that the major increase in the activities were due to the higher kcat values, indicating the Tyr->Pro mutagenesis mainly accellrated the catalysis of the enzyme but not substrate binding process. However, a 1/2 decrease in Km for L-AAFR-AMC also showed that this mutant was more favourable for bigger substrate binding. Molecular docking results also confirmed our deduction. Interupting the main chain hydrogen bond and eliminating the side chain benzyl of Tyr120 (Tyr->Pro) made it easier for substrate approach active site Cys100 and oxyanion hole Gly70, thereby increasing the enzyme catalysis. In the meanwhile, the mutant enlarged the binding entrance, thereby accelerating the substrate binding.
In conclusion, we have successfully obtained the recombined thermophilic protease PhpI. Baced on the biochemical characterization and the analysis of the 3D structure, we identified the importance of the position120 which had a close relationship with the catalysis and the substrate specificity. Our study should be useful for the rational design of new enzymes.
引文
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