植物磷酸烯醇式丙酮酸羧化酶的研究——Ⅰ.PEP羧化酶同功酶的分离和变构特性的比较

本文报导高梁(C_4植物)和小麦(C_3)植物绿色和黄化叶片中PEP羧化酶的一些特性的比较研究。结果表明不同材料叶片的PEP羧化酶对一些代谢物的反应不同。高梁绿色叶片的PEP羧化酶为G6P、Gly和FDP所激活,为油酸和柠檬酸所抑制。G6P、Gly和FDP对小麦叶片(绿色和黄化叶)、高粱黄化叶片的PEP羧化酶则均无激活作用,油酸和柠檬酸的抑制效应也消失或者下降。 比较这些不同来源的PEP羧化酶在DEAE——纤维素柱层析的结果表明它们具有不同的离子特性。在高粱绿色叶片中分得两种具有不同物理学和动力学特性的PEP羧化酶同功酶(PCⅠ;PCⅡ)。它们的Km(PEP)值各为1.66毫克分子和0.181毫克分子。PCⅡ对G6P的反应较迟钝。从NaCl洗脱梯度、聚丙烯酰胺凝胶电泳和对变构效应剂的反应来看,PCⅡ的一些特性接近于小麦的PE羧化酶。     

植物磷酸烯醇式丙酮酸羧化酶的研究——Ⅱ.高粱叶片PEP羧化酶的代谢物调节特性和油酸抑制效应

本文报导高粱叶片的PEP羧化酶与一些代谢物相互作用的动力学特性。MgCl_2对不同PEP羧化酶同工酶表现程度不同的负协同性,Hill系数分别为0`86(PC Ⅰ)和0.47(PCⅡ)。PEP的饱和曲线呈S型。Hill系数为2.4,表现为正协同性。在不同浓度的G6P存在下,曲线的S型特性消失,Hill系数下降至1。而在不同浓度甘氨酸存在下负协同程度逐步增强,Hill系数为0.72。测定不同浓度G6P对酶活化程度的影响结果表明高浓度G6P(10 mM以上)活化程度反而下降,同时加入低浓度的甘氨酸(0.1～5 mM)能减缓高浓度G6P活化作用下降的程度。上述结果表明Mg~( )和PEP不仅作为底物或辅因子参与反应而且以同位协同的方式调节酶构象的变化,G6P和gly活化酶的作用类型是不同的。低浓度油酸(5～50 μM)对酶有强烈的抑制效应。高浓度Mg~( )不能解除其对酶的抑制。不同材料的酶对油酸反应不同。使高梁叶片PC Ⅰ活性完全抑制的油酸浓度(100 μM),对PCⅡ和小麦的PEP羧化酶活性几乎没有多大影响,表明油酸对高梁光合型PEP羧化酶的选择性抑制与Mg~( )的螯合作用无关。酶先后与Mg~( )或油酸预保温试验结果表明油酸可能作用于Mg~( )在酶蛋白上的调节位置。     

马齿苋叶片PEP羧化酶的分子特性

马齿苋叶片PEPCase由四个相同的亚基组成,亚基分子量为83kD。远紫外CD光谱分析表明,此酶含有36.6％α—螺旋结构。马齿苋叶片PEPCase可被G6P激活,但不能被Gly、Ser激活。G6P可防止酶的尿素变性和枯草杆菌蛋白酶的作用。这种保护效应与G6P诱导的酶构象变化有关。 从酶对低温、高温及尿素的反应来看,马齿苋叶片PEPCase的稳定性高于高粱叶片PEP—Case,两者的免疫特性和电泳特性亦不同。     

植物磷酸烯醇式丙酮酸羧化酶的研究——Ⅲ.高粱叶片PEP羧化酶的多构象状态

应用NEM对酶的化学修饰技术,报导了半胱氨酸残基与高粱叶片的PEP羧化酶催化功能的关系。结果表明,NEM修饰使PEP羧化酶活性丧失。酶的失活速度表现为拟一级反应动力学特性。随NEM浓度的增加酶的失活速度加快。不同效应剂对酶的NEM失活具不同影响。油酸和MgCl_2促进酶的失活,G6P、甘氨酸和苹果酸各具有不同程度的保护作用。以P_(0.5)值来比较G6P和甘氨酸的保护效果,其值各为4.17mM和3.44mM。而当这两种效应剂以等量浓度同时存在时,P_(0.5)值下降为0.06mM,表现出它们的协同保护作用。从复合效应剂对酶的热失活速度和最大反应速度(V_(max))的影响亦可看出这种协同作用的存在。上述两方面的结果表明G6P和甘氨酸同时存在时诱发的酶的构象状态与它们分别存在时诱发的构象状态各不相同。根据这些结果提出了高粱叶片的PEP羧化酶可能存在的多构象状态模型,并对其生理意义进行了讨论。     

植物磷酸烯醇式丙酮酸羧化酶的研究——Ⅶ.PEP羧化酶必需赖氨酸残基的化学修饰

本文报道纯化的高粱叶片PEP 羧化酶经氨基修饰剂TNBS 和PLP 的修饰迅速失活。酶的TNBS 失活与保温时间和抑制剂浓度呈函数关系并表现为拟一级反应的特性。动力学资料表明酶仅被1分子TNBS 修饰即失活。TNBS 修饰酶的吸收光谱特性表明被修饰的是酶蛋白的赖氨酸残基。底物(PEP)和效应剂(G6P)保护酶免被TNBS 失活。计算G6P 和酶的解离常数K_d-2.39×10~(-3)M。酶的其他反应组分HCO_3~-和MgCl_2单独存在时均不影响TNBS 对酶的失活作用。在被TNBS 修饰过程中还导致酶对G6P 迅速脱敏,同时却保持酶对甘氨酸的敏感性。     

植物磷酸烯醇式丙酮酸羧化酶的研究——Ⅶ.PEP羧化酶必需赖氨酸残基的化学修饰

本文报道纯化的高粱叶片PEP羧化酶经氨基修饰剂TNBS和PLP的修饰迅速失活。酶的TNBS失活与保温时间和抑制剂浓度呈函数关系并表现为拟一级反应的特性。动力学资料表明酶仅被1分子TNBS修饰即失活。TNBS修饰酶的吸收光谱特性表明被修饰的是酶蛋白的赖氨酸残基。底物(PEP)和效应剂(G6P)保护酶免被TNBS失活。计算G6P和酶的解离常数K_d=2.39×10~(-3)M。酶的其他反应组分HCO_3~-和MgCl_2单独存在时均不影响TNBS对酶的失活作用。在被TNBS修饰过程中还导致酶对G6P迅速脱敏,同时却保持酶对甘氨酸的敏感性。     

植物磷酸烯醇式两酮酸羧化酶的研究——Ⅵ.葡萄糖-6-磷酸和甘氨酸对高粱叶片PEP羧化酶的稳定效应

高粱叶片的PEP羧化酶对温度很敏感,在45℃下迅速失活。加入变构活化剂G6P或者甘氨酸对酶的稳定性没有明显的影响。但当在C6P和甘氨酸同时存在时,酶对高温的稳定性则大大提高了。PEP羧化酶在低温中也不稳定。4℃下很快失活。酶的这种冷失活现象也可为加入效应剂G6P和甘氨酸所防止。 比较一些多羟基醇类对酶的热稳定性的影响,表明它们都显著地提高酶的热稳定性。山梨醇的保护作用最强,赤藓醇次之,甘油又次之。说明保护效应与保护剂的羟基数有关。应用含有G6P、甘氨酸和甘油(GGG)的缓冲液对提高酶在纯化和贮存过程中的稳定性非常有效。     

植物磷酸烯醇式丙酮酸羧化酶的可逆胍变性

纯化的高梁叶片磷酸烯醇式丙酮酸羧化酶(PEP羧化酶)经不同浓度的盐酸胍处理变性失活后,在试验的蛋白浓度范围内,它的失活时间进程的动力学分析表明为一级反应。0.4 M盐酸胍处理25分钟后(O℃),酶的催化活性完全丧失,酶蛋白的远紫外圆二色性光谱、内源荧光光谱及免疫特异性等测定均表明酶的结构发生了深刻变化。甘油及PEP羧化酶的变构效应剂G6P和甘氨酸对酶在盐酸胍溶液中的变性作用有一定的保护效果。变性酶用复性缓冲液稀释20倍后,在最佳条件下,再经30分钟保温,酶的催化活性能恢复70％以上。G6P和甘氨酸能促进变性酶的复性,甘油亦有明显效果。随着酶活性的恢复,它的远紫外圆二色性、内源荧光及免疫特异性也随之恢复,变性酶的复性速率在常温下(25℃)比在低温下(0℃)要快得多。     

植物磷酸烯醇式丙酮酸羧化酶的研究——Ⅷ.C_4植物PEP羧化酶的光诱导形成

连续照光可使玉米和高粱黄化叶切片PEP羧化酶活性提高,同时[~3H]一亮氨酸掺入蛋白质和叶绿素的含量也增加。 应用蛋白质合成抑制剂放线菌酮、放线菌素D和光合作用抑制剂DCMU所得资料表明光刺激黄化叶片PEP羧化酶活性的提高与光合电子传递无关而与叶片蛋白质的合成有关。放线菌酮和放线菌素D强烈抑制酶的活性,同时也抑制放射性标记化合物掺入转绿叶切片中。放射性同位素标记试验表明[~3H]一亮氨酸大量掺入转绿玉米叶切片的PEP羧化酶蛋白中。 应用PEP羧化酶抗血清进行双向免疫扩散和兔疫吸附测定得到的结果亦表明玉米、高粱等C_4植物绿色叶片的PEP羧化酶的形成受光的诱导。     

水分胁迫对露花磷酸烯醇式丙酮酸羧化酶表达水平及特性的影响

水分胁迫能引起露花叶片PEP羧化酶的活力、酶蛋白和mRNA水平的提高。复水后,叶片PEP羧化酶表达量降低;茎中的PEP羧化酶在水分胁迫和恢复水分供应过程中变化情况与叶片相似,兼性CAM植物的碳代谢类型转变发生在植物的绿色组织中。露花叶片中除了250kD的PEP羧化酶同功酶外,还有300kD同功酶;主茎的叶片叶位越低,PEP羧化酶活力越高。     

高粱叶片磷酸烯醇式丙酮酸羧化酶溶液构象状态的紫外差示吸收光谱研究

PEP诱导产生的差光谱在237nm是一强负峰,在252nm附近呈宽负峰。Mg~(2+)产生的差光谱在275nm附近为正的阔峰,在237nm处为一负峰。PEP、Mg~(2+)共同与酶作用的差光谱在263nm附近呈宽的负峰。正效应剂G6P、Gly及GG分别存在条件下PEP羧化酶的差光谱亦各具明显差异,在270nm以下光区内尤其显著。在284nm和291nm为两个负峰,Gly诱导的峰强度大于G6P的,而GG复合效应剂对此两峰的影响表现很大的协同作用。Mal作用于酶的差光谱在246nm处有一负峰。     

Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state

Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were performed with highly purified, recombinant pea NADPH:protochlorophyllide oxidoreductase (POR) heterologously expressed in Escherichia coli. The results obtained with an individual stereoisomer of the substrate [C8-ethyl-C13(2)-(R)-protochlorophyllide] demonstrate that the enzyme photoactive state possesses a characteristic fluorescence maximum at 646 nm that is due to the presence of specific charged amino acids in the enzyme catalytic site. The photoactive state is converted directly into an intermediate having fluorescence at 685 nm in a reaction involving direct hydrogen transfer from the cofactor (NADPH). Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme. At the same time, the presence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation. Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme. In addition, the presence of Tyr275 is absolutely necessary for the second step of the protochlorophyllide reduction reaction, "dark" conversion of the 685 nm fluorescence intermediate and the formation of the final product, chlorophyllide. We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state.     

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.     

Effects of stabilizing solutes on salt activation of phosphoenolpyruvate carboxylase from various plant sources

Phosphoenolpyruvate carboxylase (PEP carboxylase EC 4.1.1.31) was extracted from various halophytic, semi-halophytic and glycophytic plant species. When the enzyme of those extracts was substrate protected, and in the presence of 1.6 m M PEP in the reaction mixture, the activity of PEP carboxylase was increased by 100 m M NaCl, and the activity range in the presence of NaCl was expanded. No correlation could be established between the response of the enzyme to ions and various plant characteristics, such as taxonomic status, salt tolerance or carbon fixation pathways. Salt activation of PEP carboxylase was substrate (PEP) dependent, but the minimal substrate concentration varied in different species.
Effects of the stabilizing solutes PEP, betaine, proline and glycerol on the kinetic properties of PEP carboxylase from Zea mays (L.) cv. Hazera were analyzed. In the absence of NaCl the slope of the Hill plot (n_It) tended to rise in the presence of these solutes. Stabilization of the enzyme with betaine or glycerol caused a decrease in K'. while K' and VTO increased in the presence of PEP. NaCl (100 mM) caused an increase in both K' and V^max in the protected as well as in the unprotected enzyme, except for PEP protection, where K' decreased somewhat. In the presence of the protectants, glycerol and PEP, the effect of NaCl on V^max, was 2–4 times higher than its effect on the non-protected enzyme.     

诺卡氏菌形放线菌β-甘露聚糖酶的化学修饰及活性中心的研究

分别用 PCMB、NEM、N- AI、NBS等对诺卡氏菌形放线菌β- D-甘露聚糖酶进行化学修饰 ,证明蛋白上的巯基、酪氨酸残基及色氨酸残基是维持酶活性的必需基团 .在加入少量底物后 ,β- D-甘露聚糖酶的最大荧光发射峰从天然状态下的 336nm处蓝移至 332 nm,且峰强度有所增大 .这表明其色氨酸残基隐藏在蛋白内部的疏水区域 .通过对该酶圆二色性扫描光谱的分析 ,表明蛋白内部有二硫键的存在 ;通过巯基乙醇化学修饰的研究 ,表明二硫键是影响该酶热稳定性的一个重要因素 .在蛋白的各种二级结构中 ,α-螺旋、β-折叠、β-转角、自由卷曲的比例分别为 1 6.6%、2 5.4%、2 0 .5%和 37.5% .     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

高粱叶片磷酸烯醇式丙酮酸羧化酶疏水微区的荧光研究

应用疏水荧光探针──ＡＮＳ在不同浓度的变构效应剂存在时进行荧光滴定．磷酸烯醇式丙酮酸羧化酶分子中疏水微区的微环境能被效应剂或底物诱导产生变构,不同的变构效应剂所诱发的构象态是不一致的。这进一步证明了高粱叶片磷酸烯醇式丙酮酸羧化酶在溶液中的多构象态。     

Fluorescence spectra of luteinizing hormone releasing hormone,a decapeptide containing histidyl,tryptophyl and tyrosyl residues: Analysis by derivative spectroscopy

Fluorescence spectra have been obtained for luteinizing hormone releasing hormone, a decapeptide containing His, Trp and Tyr, and analogs lacking one or more of these residues. The second derivatives of these spectra were used to examine the contributions of the three residues to the spectrum of the hormone. Tyr influences the excitation spectrum when fluorescence is monitored at an emission wavelength of 305 nm but makes little or no contribution to the emission spectrum when the compound is excited at 275 nm. His and Trp influence both excitation and emission spectra.     

Lima bean proteinase inhibitor. Origins of circular dichroism bands and modification by Br2- and (CNS)2-.

Origins of CD bands in lima bean proteinase inhibitor were deduced from an acetylation-deacetylation study of the sole tyrosyl residue in the protein (Tyr 69), and by analogy with Bowman-Birk soybean proteinase inhibitor, a homologous protein with similar spectral properties. Tyr 69 is relatively inaccessible to N-acetylimidazole; 100-fold molar excess of the reagent in the presence of 6 M guanidine hydrochloride elicited about 70 to 80% O-acetylation. A broad negative CD band centered around 280 nm arises mainly from the longest wavelength transition of cystinyl side chains (epsilon L--epsilon R approximately equal to -0.8 M-1 cm-1 per disulfide). The second cystinyl transition gives rise to a positive CD band of a comparable intensity at 247 nm. The Lb vibronic transition of Tyr 69 has negative CD around 280 nm, contributing approximately 10% of the total CD intensity at 278 nm (epsilon L--epsilon R approximately equal to -0.5 M-1 cm-1). The 232 nm positive shoulder is from the La vibronic transition of Tyr 69. Radical anions, Br2- and (CNS)2-, generated by the irradiation of N2O-saturated inhibitor solutions containing KBr or KCNS, reduced tyrosyl CD without affecting disulfide CD bands, indicating that the radical anions damaged Tyr 69 without altering protein conformation. The inhibitor modified at Tyr 69 by Br2- and (CNS)2- retained full activity toward trypsin and chymotrypsin. The irradiation of the inhibitor in the air-saturated solution led to loss in tyrosyl as well as cystinyl CD bands and decline in both antiproteinase activities.     

Changes in Sensitivity to Effectors of Maize Leaf Phosphoenolypyruvate Carboxylase during Light/Dark Transitions

Illumination of previously darkened maize (Zea mays L. cv Golden Cross Bantam T51) leaves had no effect on the concentration of phosphoenolpyruvate (PEP) carboxylase protein, but increased enzyme activity about 2-fold when assayed under suboptimal conditions (pH 7.0 and limiting PEP). In addition, sensitivity to effectors of PEP carboxylase activity was significantly altered; e.g. malate inhibition was reduced and glucose-6-phosphate activation was increased. Consequently, 10- to 20-fold differences in PEP carboxylase activity were observed during dark to light transitions when assayed in the presence of effectors. At pH 7.0 activity of purified PEP carboxylase was not proportional to enzyme concentrations. Below 0.7 microgram PEP carboxylase protein per milliliter, enzyme activity was disproportionately reduced. Including polyethylene glycol plus potassium chloride in the reaction mixture eliminated this discontinuity and substantially increased PEP carboxylase activity and reduced malate inhibition dramatically. Inclusion of polyethylene glycol in the assay mixture specifically increased the activity of PEP carboxylase extracted from dark leaves, and reduced malate inhibition of the enzyme from both light and dark leaves. Collectively, the results suggest that PEP carboxylase in maize leaves is subjected to some type of protein modification that affects both activity and effector sensitivity. We postulate that changes in quaternary structure (dissociation or altered subunit interactions) may be involved.