菓菠萝蛋白酶的分子构象与活力变化的研究

发现CBZ-Lys·pNP能有效地被菓菠萝蛋白酶(Fruit Bromelain E.C.3.4.22.5)作用,测得Km为4.167×10~(-4)mol/L,k_(cat)为742min~(-1)。以荧光和紫外差示光谱为监测手段,对酶分子构象变化进行研究。酶的荧光强度随胍浓度增大而逐渐下降,4mol/L胍变性时,发射峰自332nm红移到353nm,并在310nm处出现新的发射峰。酶的荧光强度都因SDS存在而下降,SDS浓度大于3.47mmol/L有所回升,并出现红移,同时在315nm处出现新的发射肩;紫外差示光谱显示在236nm有一个较显著的员峰,此峰与β-螺旋结构变化有关,278、286和295nm出现三个负峰,260nm有较小正峰,说明酶分子中Tyr、Trp和Phe的微环境发生了明显的变化。测定酶在不同浓度胍和SDS中的变性和失活速度常数,对酶构象变化及催化活力的关系作了比较研究,酶的失活速度均大于变性速度。     

Inactivation Kinetics of Guanidinium Chloride on <Emphasis Type="Italic">Penaeus vannamei</Emphasis>β - <Emphasis Type="Italic">N</Emphasis>-Acetyl-D-Glucosaminidase and the Relationship of Enzyme Activity and its Conformation

The effects of guanidinium chloride (GuHCl) on the activity of Penaeus vannamei β-N-acetyl-d-glucosaminidase (NAGase) have been studied. The results show that GuHCl, at appropriate concentrations, can lead to reversible inactivation of the enzyme, and the IC₅₀ is estimated to be 0.6 M. Changes of activity and conformation of the enzyme in different concentrations of GuHCl have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreases distinctly with increasing GuHCl concentrations, and the emission peaks appear red-shifted (from 339.4 to 360 nm). Changes in the conformation and catalytic activity of the enzyme are compared. The extent of inactivation is greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. The kinetics of inactivation has been studied using the kinetic method of the substrate reaction. The rate constants of inactivation have been determined. The value of k₊₀ is larger than that of k₊₀^′ which suggests that the enzyme is protected by substrate to a certain extent during guanidine denaturation.     

Conformational and activity changes during guanidine denaturation of D-glyceraldehyde-3-phosphate dehydrogenase

Changes in intrinsic protein fluorescence of lobster muscle D-glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been compared with inactivation of the enzyme during denaturation in guanidine solutions. The holoenzyme is completely inactivated at guanidine concentrations less than 0.5 M and this is accompanied by a red shift of the emission maximum at 335 nm and a marked decrease in intensity of the intrinsic fluorescence. At 0.5 M guanidine, the inactivation is a slow process, with a first-order rate constant of 2.4 X 10(-3) s-1. A further red shift in the emission maximum and a decrease in intensity occur at guanidine concentrations higher than 1.5 M. The emission peak at 410 nm of the fluorescent NAD derivative introduced at the active site of this enzyme (Tsou, C.L. et al. (1983) Biochem. Soc. Trans. 11, 425-429) shows both a red shift and a marked decrease in intensity at the same guanidine concentration required to bring about the inactivation and the initial changes in the intrinsic fluorescence of the holoenzyme. It appears that treatment by low guanidine concentrations leads to both complete inactivation and perturbation of the active site conformation and that a tryptophan residue is situated at or near the active site.     

α-淀粉酶在盐酸胍和碳酸胍变性中有关胍基作用的研究

利用紫外差谱、荧光光谱和园二色谱法对比地研究了α-淀粉酶盐酸胍和碳酸胍变性,分析了两种胍变性明显差异的原因。通过等同的胍基浓度下,α-淀粉酶两种胍变性的构象变化与活性关系的实验,表明同等摩尔浓度的两种胍盐变性能力上的明显差异并不主要是由于它们胍基含量上的不同。将盐酸胍从中性pH(6.5)调至碱性pH(10.4),其变性能力大增,紫外差谱与碳酸胍变性相似,出现了290nm的正肩和296nm的正峰,与此同时,酶的荧光强度大大降低,大部分酶活性丧失。由此推论,两种胍变性能力的明显差异的重要原因之一是在碱性介质中胍基的变性能力明显增强,并分析了其增强的原因。     

Comparison of inactivation and unfolding of methanol dehydrogenase during denaturation in guanidine hydrochloride and urea

The activity and the conformational changes of methanol dehydrogenase (MDH), a quinoprotein containing pyrrolo-quinoline quinone as its prosthetic group, have been studied during denaturation in guanidine hydrochloride (GdnHCl) and urea. The unfolding of MDH was followed using the steady-state and time resolved fluorescence methods. Increasing the denaturant concentration in the denatured system significantly enhanced the inactivation and unfolding of MDH. The enzyme was completely inactivated at 1 M GdnHCl or 6 M urea. The fluorescence emission maximum of the native enzyme was at 332 nm. With increasing denaturant concentrations, the fluorescence emission maximum red-shifted in magnitude to a maximum value (355 nm) at 5 M GdnHCl or 8 M urea. Comparison of inactivation and conformational changes during denaturation showed that in general accord with the suggestion made previously by Tsou, the active sites of MDH are situated in a region more flexible than the molecule as a whole.     

酵母乙醇脱氢酶胍变性时的失活与去折叠的比较研究

应用荧光发射光谱,圆二色光谱,二阶导数光谱和紫外差吸收光谱等监测手段,研究了酵母乙醇脱氢酶在胍溶液中的去折叠。比较不同盐酸胍浓度下酵母乙醇脱氢酶的失活与构象变化,实验表明酶的失活先于构象变化：在低浓度胍溶液中,构象尚未发生明显变化时,酶活几乎已经完全丧失。由上述结果可见,含有辅基金属离子Ｚｎ~（２＋）酶的活性部位较酶分子的整体结构也具有柔性。     

无花果蛋白酶在胍溶液中的分子折叠与活力变化研究

本文研究无花果蛋白酶（ＥＣ．３．４．４．１２）在不同浓度盐酸胍溶液中分子构象与活力变化关系。酶的内源荧光光谱,圆二色光谱与酶活力的变化表明：荧光光谱呈现二个明显的变化区域,低浓度胍（低于２ｍｏｌ／Ｌ）中,荧光发射峰基本不变,但荧光强度随胍浓度上升,随胍浓度断续增大（高于２ｍｏｌ／Ｌ）,酶的最大发射波长明显红移。当胍浓度低于１ｍｏｌ／Ｌ时,不仅不会使酶失活,反而使酶激活,当胍浓度高于１ｍｏｌ／Ｌ以上时,酶逐渐失活,使酶完全失活的胍浓度为６ｍｏｌ／Ｌ酶的圆二色光谱也随着胍浓度的改变而发生复杂的变化。将荧光变化,ＣＤ谱变化及活力改变结合起来,表明活力的激活与构象的明显变化似是同步发生的,从另一角度进一步说明酶活性部位柔性是充分表现酶活力所必需。     

SNase R在胍变性过程中构象与活力变化的比较

以紫外差光谱、荧光光谱为监测手段对金黄色葡萄球菌核酸酶类似物(SNase R)在胍溶液中构象与活力变化进行了比较.SNase R在Llmol L0.8mol L和0.5mol L胍溶液变性时变性过程均为两个一级反应,但是酶在上述胍浓度下失活的速度远快于构象变化的速度:酶在同一胍浓度下活力丧失的程度也远快于构象变化的程度.上述结果表明:SNase R的活性部位可能位于柔性较大的区域.     

根霉葡萄糖淀粉酶的复性复活动力学研究

本文利用荧光、紫外差光谱研究了根霉葡萄糖淀粉酶在盐酸胍变性后的复性、复活动力学。结果表明,该酶在小于4mol/L盐酸胍中变性是可逆的,其复性过程遵循一级反应方程。酶复活过程是由两个一级反应组成的复合反应,构象变化速度与复活过程中较快的反应速度相差无几,这可能是在Trp及Tyr微区的构象变化基本完成之后,酶活力恢复还没有完成造成的。     

The refolding of denatured rabbit muscle pyruvate kinase.

The refolding of rabbit muscle pyruvate kinase after denaturation by guanidine hydrochloride was studied. On dilution of the denaturing agent, enzyme activity is only partially regained. The extent of regain of activity is dependent on protein concentration, showing a marked decrease at higher concentrations. The failure to regain complete activity appears to be related to the formation of inactive aggregates, which can be separated from active enzyme by gel filtration. Insoluble aggregates can be partially re-activated after solubilization in guanidine hydrochloride. Changes in the circular-dichroism and fluorescence spectra during refolding suggest that a partially folded, inactive species is formed rapidly; this differs from native enzyme in being more susceptible to proteolysis by trypsin.     

截流荧光法研究米曲霉氨基酰化酶的快速胍变性动力学

 用荧光光谱法、截流荧光法和酶活力测定法研究了在盐酸胍溶液中米曲霉氨基酰化酶变性动力学。我们发现在4.8mol/L盐酸胍溶液作用下(0.05mol/L磷酸缓冲溶液,pH7.4,25℃),氨基酰化酶二聚体解离成单亚基过程是一个十分快速的过程,反应速率常数k为3361l/s,即约需3ms时间完成;而单亚基分子的构象变化需要约20min方能到达平衡态,这是一个逐渐变化的缓慢过程。酶分子在胍作用下的失活现象同酶分子的结构变化紧密相关,在胍浓度大于4mol/L时酶完全失活。在高浓度盐酸胍下酶失活主要是因为酶二聚体迅速解离成单亚基的过程和单亚基构象逐渐变化的缓慢过程。双亚基解离常数大小标志着酶分子亚基间作用力的强弱。     

Effects of arginine on rabbit muscle creatine kinase and salt-induced molten globule-like state

The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H(2)O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK.     

Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable. Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 +/- 1500 for both native and denatured enzyme. The unfolding transition at varying guanidine X HCl concentrations is characterized by a two-step profile: at 0.4-0.8 M, partial unfolding is parallelled by inactivation; at 2.0-2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4-1.8 M guanidine X HCl, partial unfolding is superimposed by aggregation. The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine X HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%. Denaturation in 6 M guanidine X HCl, as monitored by the decrease of protein fluorescence, is fast (less than 8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (less than 20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long-term denaturation, is determined by a single first-order reaction characterized by tau = 29 +/- 3 min (20 degrees C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate-limiting for reactivation after long-term denaturation, are characterized by a high energy of activation (112 +/- 8 kJ/mol). The slow reactivation step is compatible in rate with the first-order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62-90].     

Unfolding and inactivation during thermal denaturation of an enzyme that exhibits phytase and acid phosphatase activities

The thermostability of an enzyme that exhibits phytase and acid phosphatase activities was studied. Kinetics of inactivation and unfolding during thermal denaturation of the enzyme were compared. The loss of phytase activity on thermal denaturation is most suggestive of a reversible process. As for acid phosphatase activities, an interesting phenomenon was observed; there are two phases in thermal inactivation: when the temperature was between 45 and 50 degrees C, the thermal inactivation could be characterized as an irreversible inactivation which had some residual activity and when the temperature was above 55 degrees C, the thermal inactivation could be characterized as an irreversible process which had no residual activity. The microscopic rate constants for the free enzyme and substrate-enzyme complex were determined by Tsou's method [Adv. Enzymol. Relat. Areas Mol. Biol. 61 (1988) 381]. Fluorescence analyses indicate that when the enzyme was treated at temperatures below 60 degrees C for 60 min, the conformation of the enzyme had no detectable change; when the temperatures were above 60 degrees C, some fluorescence red-shift could be observed with a decrease in emission intensity. The inactivation rates (k(+0)) of free enzymes were faster than those of conformational changes during thermal denaturation at the same temperature. The rapid inactivation and slow conformational changes of phytase during thermal denaturation suggest that inactivation occurs before significant conformational changes of the enzyme, and the active site of this enzyme is situated in a relatively fragile region which makes the active site more flexible than the molecule as a whole.     

Comparison of the rates of inactivation and conformational changes of creatine kinase during urea denaturation

The denaturation of creatine kinase in urea solutions of different concentrations has been studied by following the changes in the ultraviolet absorbance and intrinsic fluorescence as well as by the exposure of hidden SH groups. In concentrated urea solutions, the denaturation of the enzyme results in negative peaks at 285 nm with shoulders at 280 and 290 nm and positive peaks at 244 and 302 nm in the denatured minus native enzyme difference spectrum. The fluorescence emission maximum of the enzyme red shifts with increasing intensity in urea solutions of increasing concentrations. At least part of these changes can be attributed to direct effects of urea on the exposed Tyr and Trp residues as shown by experiments with model compounds. The inactivation of this enzyme has been followed and compared with the conformational changes observed during urea denaturation. A marked decrease in enzyme activity is already evident at low urea concentrations before significant conformational changes can be detected by the exposure of hidden SH groups or by ultraviolet absorbance and fluorescence changes. At higher urea concentrations, the enzyme is inactivated at rates 3 orders of magnitude faster than the rates of conformational changes. The above results are in accord with those reported previously for guanidine denaturation of this enzyme [Yao, Q., Hou, L., Zhou, H., & Tsou, C.-L. (1982) Sci. Sin. (Engl. Ed.) 25, 1186-1193] and can best be explained by assuming that the active site of this enzyme is situated near the surface of the enzyme molecule and is sensitive to very slight conformational changes.     

Conformational changes and inactivation of rabbit muscle creatine kinase in dimethyl sulfoxide solutions.

The effects of dimethyl sulfoxide (DMSO) on creatine kinase (CK) conformation and enzymatic activity were studied by measuring activity changes, aggregation, and fluorescence spectra. The results showed that at low concentrations (< 65% v/v), DMSO had little effect on CK activity and structure. However, higher concentrations of DMSO led to CK inactivation, partial unfolding, and exposure of hydrophobic surfaces and thiol groups. DMSO caused aggregation during CK denaturation. A 75% DMSO concentration induced the most significant aggregation of CK. The CK inactivation and unfolding kinetics were single phase. The unfolding of CK was an irreversible process in the DMSO solutions. The results suggest that to a certain extent, an enzyme can maintain catalytic activity and conformation in water-organic mixture environments. Higher concentrations of DMSO affected the enzyme structure but not its active site. Inactivation occurred along with noticeable conformational change during CK denaturation. The inactivation and unfolding of CK in DMSO solutions differed from other denaturants such as guanidine, urea, and sodium dodecyl sulfate. The exposure of hydrophobic surfaces was a primary reason for the protein aggregation.     

淀粉液化芽孢杆菌α-淀粉酶在盐酸胍和碳酸胍变性过程中的构象变化

 利用紫外差光谱,荧光光谱和圆二色谱法对比地研究了淀粉液化茅孢杆菌α-淀粉酶在盐酸胍和碳酸胍变性过程的构象变化与活性关系以及在变性早期钙离子对酶构象的稳定作用。     

脲对果菠萝蛋白酶活力与构象的影响

本文用荧光光谱,紫外差示光谱和CD谱研究果菠萝蛋白酶在不同浓度的脲溶液中的构象及酶活力的变化情况。酶的荧光强度随脲浓度增大而明显增加,8mol/L脲使荧光强度增强65％,发射峰出现红移。差示谱表明在232nm和288nm出现二个正峰,它们均随脲浓度增大而加剧,前者与主链构象变化有关,而后者与生色基团(Trp、Tyr)的微环境变化相关。CD谱表明:天然酶在208nm和225nm处有二个负峰,脲变性后,225nm的负峰基本上不随脲浓度增大而变化,但208nm峰则明显发生变化并逐渐出现红移,6mol/L以上此峰则完全消失。     

缢蛏碱性磷酸酶胍溶液中分子折叠与活力变化研究

报道了缢蛏碱性磷酸酶（简称ＡＬＰ）经不同浓度盐酸胍处理时酶的分子构象所发生的变化以及酶变化和失活的动力学过程。在胍中酶荧光发射峰强度下降,紫外差光谱在２４６ｎｍ和２８５ｎｍ处出现２个负峰,ＣＤ谱中酶的α螺旋度下降,且随浓度增大,变化程度也加大。动力学研究表明,酶在０．５ｍｏｌ／Ｌ、１．０ｍｏｌ／Ｌ、２．０ｍｏｌ／Ｌ３．０ｍｏｌ／Ｌ、４．０ｍｏｌ／Ｌ盐酸胍中的变性速度常数分别为３．２１×１０~（－４）ｓ~（－１）、６．３８×１０~（－４）ｓ~（－１）、２．１７×１０~（－３）ｓ~（－１）、２．３３×１０~（－３）ｓ~９－１）、５．１７×１０~（－３）ｓ~（－１）;而酶在相应盐酸胍中的失活速度常数分别为２．３３×１０~（－４）ｓ~（－１）、３．５７×１０~（－４）ｓ~（－１）、５．８６×１０~（－４）ｓ~（－１）、１．１４×１０~（－３）ｓ~（－１）、３．４５×１０~（－３）ｓ~（－１）;表现为失活与构象伸展变化基本平行。     

Inhibition kinetics of hydrogen peroxide on beta-n-acetyl-D-glucosaminidase from prawn (Penaeus vannamei)

The effects of hydrogen peroxide (H2O2) on prawn NAGase activity for the hydrolysis of pNP-beta-D-GlcNAc have been studied. The results show that H2O2 can reversible inhibit the enzyme (IC50 = 0.85 M) and the inhibition is of a mixed type. The kinetics show that k+o is much larger than k+0, indicating the free enzyme is more susceptible than the enzyme-substrate complex in the H2O2 solution. It is suggested that the presence of the substrate offers marked protection against inhibition by H202. Changes of activity and conformation of the enzyme in different concentrations of H202 have been compared by measuring the fluorescence spectra and residual activity and show that the change of conformation is more rapidly than that of the residual activity, which implies that the whole conformation of the enzyme changes more rapidly than the conformation of the active centre of the enzyme in the H2O2 solution.