氨基酰化酶在LDS溶液中的失活与去折叠的比较研究

氨基酰化酶在阴离子去圬剂十二烷基硫酸锂（ＬＤＳ）溶液中的失活与去折叠的研究结果表明,在低浓度的ＬＤＳ溶液（０．６ｍｍｏｌ／Ｌ）中变性时,以荧光和紫外差吸收方法监测的酶分子构象尚未发生明显变化。而酶的活力已经大部分或几乎全部丧失。当ＬＤＳ浓度达１．６ｍｍｏｌ／Ｌ时,此时酶分子的构象变化才达到最大程度,在实验使用的ＬＤＳ的浓度范围内,用远紫外ＣＤ光谱监测的二级结构没有发生明显的变化。从上述研究结果,可以认为含锌氨基酰化酶的活性部位也具有相对的柔性。     

Inactivation and unfolding of aminoacylase during denaturation in sodium dodecyl sulfate solutions

During denaturation by sodium dodecyl sulfate (SDS), aminoacylase shows a rapid decrease in activity with increasing concentration of the detergent to reach complete inactivation at 1.0 mM SDS. The denatured minus native-enzyme difference spectrum showed two negative peaks at 287 and 295 nm. With the increase of concentration of SDS, both negative peaks increased in magnitude to reach maximal values at 5.0 mM SDS. The fluorescence emission intensity of the enzyme decreased, whereas there was no red shift of emission maximum in SDS solutions of increasing concentration. In the SDS concentration regions employed in the present study, no marked changes of secondary structure of the enzyme have been observed by following the changes in far-ultraviolet CD spectra. The inactivation of this enzyme has been followed and compared with the unfolding observed during denaturation in SDS solutions. A marked inactivation is already evident at low SDS concentration before significant conformational changes can be detected by ultraviolet absorbance and fluorescence changes. The inactivation rate constants of free enzyme and substrate-enzyme complex were determined by the kinetics method of the substrate reaction in the presence of inactivator previously described by Tsou [Tsou (1988),Adv. Enzymol. Related Areas Mol. Biol. 61, 381–436]. It was found that substrate protects against inactivation and at the same SDS concentrations, the inactivation rate of the free enzyme is much higher than the unfolding rate. The above results show that the active sites of metal enzyme containing Zn²⁺ are also situated in a limited and flexible region of the enzyme molecule that is more fragile to denaturants than the protein as a whole.     

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.     

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。     

Activation and conformational changes of adenylate kinase in urea solution

Ａｄｅｎｙｌａｔｅｋｉｎａｓｅ(ＥＣ2.7.4.3)ｃａｔａｌｙｚｅｓｔｈｅｉｎｔｅｒｃｏｎｖｅｒｓｉｏｎｏｆａｄｅｎｉｎｅｎｕｃｌｅｏｔｉｄｅｓａｃｃｏｒｄｉｎｇｔｏ:ＡＤＰ+ＭｇＡＤＰＡＭＰ+ＭｇＡＴＰ.Ｉｔｉｓｕｂｉｑｕｉｔｏｕｓａｎｄｐａｒｔｉｃｕｌａｒｌｙａｂｕｎｄａｎｔｉｎｔｉｓｓｕｅｓｗｉｔｈｈｉｇｈｅｎｅｒｇｙｔｕｒｎｏｖｅｒ.Ｒａｂｂｉｔｍｕｓｃｌｅａｄｅｎｙｌａｔｅｋｉｎａｓｅｉｓａｍｏｎｏｍｅｒｅｎｚｙｍｅｗｉｔｈｔｗｏｓｔｒｕｃｔｕｒｅｄｏｍａｉｎｓｃｏｍｐｏｓｅｄｏｆ194ａｍｉｎｏａ…     

人肌肌酸激酶胍变性时的失活与构象变化的比较研究

应用二阶导数光谱、紫外差吸收光谱和荧光光谱等监测手段,研究了人肌肌酸激酶在盐酸胍溶液中的构象变化。二阶导数光谱结果表明,若以6M盐酸胍中肌酸激酶酪氨酸残基的暴露程度为100％,则天然酶酪氨酸残基的暴露程度只有2％。而紫外差吸收光谱和荧光光谱的变化与兔肌肌酸激酶的结果相似。比较不同胍浓度下人肌肌酸激酶的失活与构象变化,表明酶的失活先于构象变化。同时还测定了不同浓度胍溶液中人肌酶的失活与构象变化的速度常数。结果表明以几种方法测定的构象变化均为单相的一级过程,而酶的失活却呈现了由快慢两相组成的一级反应过程。比较同浓度胍溶液中的失活速度与构象变化速度,发现酶失活的快相反应速度常数比构象变化的速度常数大1—2个数量级,慢相速度常数与构象变化速度常数相近。上述结果进一步支持了酶的活性部位构象柔性的观点。     

Effect of ethanol on the activity and conformation of Penaeus penicillatus acid phosphatase.

The effect of ethanol on the activity of Penaeus penicillatus acid phosphatase has been studied. The results show that ethanol significantly inhibits enzyme activity as a non-competitive inhibitor, with Ki 8.75%. The conformational changes of the enzyme molecule induced by ethanol were followed using fluorescence emission, ultraviolet difference and circular dichroism (CD) spectra. Increasing the ethanol concentration caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with ethanol had two negative peaks at 220 and 278 nm, and a positive peak at 240 nm. Increasing the ethanol concentration produced a small shoulder peak at 287 nm in addition to the increases in the negative magnitudes of the 220 and 278 nm peaks. The changes of the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of the tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed. The results suggest that ethanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.     

Aggregation, dissociation and unfolding of glucose dehydrogenase during urea denaturation

The effect of urea on glucose dehydrogenase from Bacillus megaterium has been studied by following changes in enzymatic activity, conformation and state of aggregation. It was found that the denaturation process involves several transitions. At very low urea concentrations (below 0.5 M), where the enzyme is fully active and tetrameric, there is a conformational change as monitored by an increase in intensity of the tryptophan fluorescence and a maximum exposure of organized hydrophobic surfaces as reported by the fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5.5'-disulfonic acid. At slightly higher urea concentrations (0.75-2 M), a major conformational transition occurs, as monitored by circular dichroism and fluorescence measurements, in which the enzyme activity is completely lost and is concomitant with the formation of interacting intermediates that lead to a highly aggregated state. Increasing urea concentrations cause a complete dissociation to lead first a partially and eventually the complete unfolded monomer. These phenomena are fully reversible by dilution of denaturant. It is concluded that after urea denaturation, the folding/assembly pathway of glucose dehydrogenase occurs with the formation of intermediate species in which transient higher aggregates appear to be involved.     

Unfolding and inactivation of Ampullarium crossean beta-glucosidase during denaturation by guanidine hydrochloride

Changes of activity and conformation of Ampullarium crossean beta-glucosidase in different concentrations of guanidine hydrochloride (GuHCl) have been studied by measuring the fluorescence spectra and its relative activity after denaturation. The fluorescence intensity of the enzyme decreased distinctly with increasing guanidine concentrations, the emission peaks appeared red shifted (from 338.4 to 350.8 nm), whereas a new fluorescence emission peak appeared near 310 nm. Changes in the conformation and catalytic activity of the enzyme were compared. A corresponding rapid decrease in catalytic activity of the enzyme was also observed. The extent of inactivation was greater than that of conformational changes, indicating that the active site of the enzyme is more flexible than the whole enzyme molecule. k(+0)>k(+0)' also showed that the enzyme was protected by substrate to a certain extent during guanidine denaturation.     

Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate

Fatty acid synthase is an important enzyme participating in energy metabolism in vivo. The inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver in SDS solutions have been studied. The results show that the denaturation of this multifunctional enzyme by SDS occurred in three stages. At low concentrations of SDS (less than 0.15 mM) the enzyme was completely inactivated with regard to the overall reaction. For each component of the enzyme, the loss of activity occurred at higher concentrations of SDS. Significant conformational changes (as indicated by the changes of the intrinsic fluorescence emission and the ultraviolet difference spectra) occurred at higher concentrations of SDS. Increasing the SDS concentration caused only slight changes of the CD spectra, indicating that SDS had no significant effect on the secondary structure of the enzyme. The results suggest that the active sites of the multifunctional fatty acid synthase display more conformational flexibility than the enzyme molecule as a whole.     

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.     

Activity change during unfolding of bovine pancreatic ribonuclease A in guanidine

Bovine pancreatic ribonuclease A loses almost completely its activity in 2-3 M guanidine, whereas only very slight conformational changes can be detected when following its unfolding by changes in its intrinsic fluorescence at 305 nm and ultraviolet absorbance at 287 nm. Reactivation on diluting out the denaturant is a time-dependent process, indicating that the inactivation is not due to inhibition by a reversible association of the enzyme with guanidine. The kinetic method of following the substrate reaction, in the presence of the denaturant previously proposed for use in the study of rapid inactivation reactions (Tian, W.X. and Tsou, C.-L. (1982) Biochemistry 21, 1028-1032), is applied to examine the inactivation rates of this enzyme during guanidine denaturation, and these have been compared with the unfolding rates as followed by fluorescence and absorbance changes. It is shown that during the unfolding of this enzyme in guanidine, the inactivation of the enzyme occurs within the dead time of mixing in a stopped-flow apparatus and is at least several orders of magnitude faster than the unfolding reaction as detected by the optical parameters. It appears that, as in the case of creatine kinase reported previously, the active site of a small enzyme stabilized by multiple disulfide linkages, such as ribonuclease A, is also situated in a region which is much more liable to being perturbed by denaturants than is the molecule as a whole.     

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。     

p-Chloromercuribenzoate-induced inactivation and partial unfolding of porcine heart lactate dehydrogenase

Purified porcine heart lactate dehydrogenase was inactivated and partially unfolded with p-chloromercuribenzoate (pCMB). With the increase of pCMB/enzyme ratio the enzyme was gradually inhibited till almost completely inactivated at the pCMB/enzyme ratio of 20 : 1. Native polyacrylamide gel electrophoresis showed that with the increase of pCMB/enzyme ratio the bands of native enzyme decreased till completely vanished. Meanwhile inactive multiple bands emerged and became thicker, which implied that lactate dehydrogenase became loose. The conformational changes of the enzyme molecule modified with pCMB were followed using fluorescence emission, ultraviolet difference, and circular dichroism (CD) spectra. Increasing pCMB concentration resulted in the decrease of fluorescence emission intensity. The ultraviolet difference spectra of the enzyme modified with pCMB exhibited an increasing absorbance in the vicinity of 240 nm with the increasing concentration of the inhibitor. The changes of the fluorescence and ultraviolet difference spectra reflected the conformational changes of the enzyme. The CD spectrum changes of the enzyme showed that its secondary structure changed as well. These results suggest that pCMB not only inhibits this enzyme but also influences its conformation (partial unfolding).     

Effect of methanol on the activity and conformation of acid phosphatase from the prawn Penaeus penicillatus

Prawn (Penaeus penicillatus) acid phosphatase (EC 3.1.3.2) catalyzes the nonspecific hydrolysis of phosphate monoesters. The effects of some pollutants in sea water on the enzyme activity results in the loss of the biological function of the enzyme, which leads to disruption of phosphate metabolism in cells. This paper analyzes the effects of methanol on the activity and conformation of prawn acid phosphatase. The results show that low concentrations of methanol can lead to reversible inactivation. Inhibition of the enzyme by methanol is classified as non-competitive inhibition, and the inhibition constant (Ki) is 8.5%. Conformational changes of the enzyme molecule in methanol solutions of different concentrations were measured using fluorescence emission, differential UV-absorption, and circular dichroism spectra. Increased methanol concentrations caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with methanol had two negative peaks, at 222 and 270 nm, and a positive peak at 236 nm. The changes in the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed some. These results suggest that methanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.     

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.     

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。     

Effect of Zinc Ions on Conformational Stability of Yeast Alcohol Dehydrogenase

Yeast alcohol dehydrogenase preparations were prepared with the conformational zinc ion removed (Apo-I YADH) and with both the conformational and catalytic zinc ions removed (Apo-II YADH). The unfolding of Apo-I YADH and Apo-II YADH during denaturation in urea solutions was then followed by fluorescence emission, circular dichroism, and second-derivative optical spectroscopies. Compared with the native enzyme, Apo-I YADH incurred some slight unfolding, and its stability against urea was markedly decreased, while Apo-II YADH incurred marked unfolding but contained residual ordered structure even at high urea concentrations. The results show that native YADH is more conformationally stable against urea denaturation than Apo-I YADH, indicating that the conformational Zn²⁺ plays an important role in stabilizing the conformation of the YADH molecule. However, unfolding of the region around the conformational zinc ion is shown not to be the rate limited step in the unfolding of the molecule by the fact that the unfolding and inactivation rate constants of native and Apo-I YADH are the same. It is suggested that the catalytic zinc ion is more important in maintaining the structure of YADH. YADH lost its cooperative unfolding ability after the zinc ions were removed. The shape of the transition curves of Apo-I YADH suggests the existence of an unfolding intermediate. For both native and Apo-I YADH, inactivation occurs at much lower urea concentrations than that needed to produce significant conformational changes of the enzyme molecule. At urea concentration above 4 M, the inactivation rate constants are much higher than those of the fast phase of the reaction of unfolding. These results support the suggestion of flexibility at the active site of the enzyme (C. L. Tsou (1986) Trends Biochem. Sci., 11, 427-429; (1993) Science, 262, 308-381).     

Unfolding and inactivation of fatty acid synthase from chicken liver during urea denaturation

The inactivation and conformational changes of the multifunctional fatty acid synthase (acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing), EC 2.3.1.85) from chicken liver have been studied in urea solution. The results show that complete inactivation of the fatty acid synthase occurs before obvious conformational changes with regard to the overall, beta-ketoacyl reduction and acetoacetyl-CoA reduction reactions. Significant conformational changes indicated by the changes of the intrinsic fluorescence emission and the circular dichroism spectra occurred at higher urea concentrations. The kinetic rate constants for the two phase inactivation and unfolding reactions were measured and semilogarithmic plots of the activity versus time gave curves which could be resolved into two straight lines, indicating that both the inactivation and unfolding processes consisted of fast and slow phases as a first-order reaction. The results from Lineweaver-Burk plots indicated that urea is a competitive inhibitor for acetyl-CoA and malonyl-CoA, with K(m) increasing with increasing urea concentrations. However, urea is a noncompetitive inhibitor for NADPH, the substrate of the overall reaction and beta-ketoacyl reduction reaction, and acetylacetate, the substrate of the beta-ketoacyl reduction reaction. Activation by low concentrations of urea was observed although this activation was only temporarily induced in an early stage of inactivation. The aggregation phenomenon of the fatty acid synthase in a certain concentration range of urea (3-4 M) was also observed during unfolding. This result shows that this multifunctional enzyme unfolds with competition with misfolding in the folding pathway. Comparison of inactivation and conformational changes of the enzyme as well as aggregation imply that unfolding intermediates may exist during urea denaturation. The possible unfolding pathway of fatty acid synthase is also discussed in this paper.     

Fluorescence studies on the dissociation and denaturation of pigeon liver malic enzyme

Exposure of pigeon liver malic enzyme [S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) in medium concentrations of guanidine-HCl at 25 degrees C and pH 7.45 caused biphasic conformational changes of the enzyme molecule. Molecular weight determination confirmed that the enzyme tetramers were dissociated to monomers in phase I transition. Enzymatic activity was completely lost in this phase. Recovery of the enzyme activity was only possible in the early stages of the phase I transition. Phase II was due to enzyme unfolding, as judged by circular dichroism and the fluorescence parameters of the enzyme. The steps of the transformation of native malic enzyme into a completely denatured state were in the following sequence: tetramer----monomer----random coil. Extensive denaturation of the enzyme molecule resulted in irreversible aggregation. Dissociation and denaturation were accompanied by a red-shift of the fluorescence spectrum (328----368 nm). Fluorescence quenching studies indicated that tryptophan residues of the enzyme molecule were buried deeply in the interior of the molecule. The tryptophan residues were only partially accessible by acrylamide and almost inaccessible by KI. Dissociation and denaturation were accompanied by exposure of the tryptophan residues, as manifested by the accessibility of the enzyme molecule toward KI or acrylamide.