四季豆幼苗尿囊酸酰胺水解酶的纯化和性质研究(简报)

从四季豆幼苗提取、部分纯化尿囊酸酰胺水解酶,分离出两个同功酶：一分子量同功酶（尿圳酸酰胺水解酶Ⅰ）,另一为小分子量同功酶（尿囊酸酰胺水解酶Ⅱ）,并对后者的性质进行研究。     

豇豆初生叶多胺氧化酶的分子特性（英文）

从6 d苗龄的豇豆幼苗初生叶中提纯得到的多胺氧化酶是一种糖蛋白,其碳水化合物含量为8.17%.全酶分子量约为146 kD,由分子量为70kD的两个相同亚基组成,每个亚基含1个Cu2+.该酶的等电点为6.2,吸收光谱分别在波长278 nm和500 nm处有1吸收峰.8种蛋白质修饰剂修饰试验并配合底物保护证实酪氨酸、赖氨酸和色氨酸残基及-SH都不是该酶活性中心的必需基团,而组氨酸残基则是活性中心的必需基团.进一步分析部分失活的修饰酶动力学参数的变化得知,组氨酸残基可能处于酶分子的催化部位而非底物的结合部位.     

豇豆初生叶多胺氧化酶的分子特性

从6d苗龄的豇豆幼苗初生叶中提纯得到的多胺氧化酶是一种糖蛋白,其碳水化合物含量为8．17％,全酶分子量约为146kD,由分子量为70kD的两个相同亚基组成,每个亚基含1个Cu^2 。该酶的等电点为6．2,吸收光谱分别在波长278nm和500nm处有1吸收峰。8种蛋白质修饰剂修饰试验并配合底物保护证实酷氨酸,赖氨酸和色氨酸残基及－SH都不是该酶活性中心的必需基团,而组氨酸残基则是活性中心的必需基团,进一步分析部分失活的修饰酶动力学参数的变化得知,组氨酸残基可能处于酶分子的催化部位而非底物的结合部位。     

菜心乙醇酸氧化酶的纯化和催化特性分析

用改进后的方法,从菜心绿叶中分离纯化得到一个亚基分子量为42kD的乙醇酸氧化酶,用氧电极法测定该酶同时能催化乙醇酸和乙醛酸的氧化。     

大肠杆菌AS1.357 L-天门冬酰胺酶的选择性化学修饰

用九种化学修饰剂研究了大肠杆菌AS1.357 L-天门冬酰胺酶分子中的五种不同氨基酸侧链基团与催化活性的关系。结果说明,渡酶活力与硫氧墓完全无关;与色氨酸、精氨酸和组氨酸亦无直接联系;而酪氨酸残基和羧基的修饰引起酶活力急剧下降。其中酪氢酸残基巳被证实是该酶活力的必需基团,处于该酶分子的活性部位。     

荞麦与大豆叶片中草酸含量差异及其可能的原因

用1/5浓度Hoagland营养液培养荞麦和大豆幼苗l0 d后,荞麦叶片、根及其分泌物中的草酸含量均明显高于大豆,说明荞麦叶片的草酸形成能力强.荞麦叶片中存在少量的草酸氧化酶活性,而大豆中未检测到该酶活性,表明荞麦具有一定的降解草酸的能力.乙醇酸氧化酶(GO)催化乙醇酸氧化的活性两种植物之间虽差异不明显,但该酶催化乙醛酸氧化的活性荞麦显著高于大豆.荞麦GO对乙醛酸的Km值明显低于大豆GO,同时其乙醛酸含量也较高,因此其叶片中由乙醛酸形成草酸的速率应高于大豆.由此认为,由乙醛酸氧化生成草酸可能是植物草酸合成限速步骤之一,其反应速率高低可能导致不同种类植物叶片中草酸含量的差异.     

菜心乙醇酸氧化酶的纯化和催化特性分析

用改进后的方法,从菜心绿叶中分离纯化得到一个亚基分子量为４２ｋＤ的乙醇酸氧化酶,用氧电极法测定该酶同时能催化乙醇酸和乙醛酸的氧化。     

组氨酸残基在乙醇酸氧化酶活性中的作用

DEPC能显著抑制GAO的活性。其失活速度表现为假一级动力学特性,并和抑制剂浓度成线性正比关系。底物乙醇酸可保护GAO免受DEPC抑制,羟胺能使被抑制的酶重新复活。光谱测定表明,被抑制的酶只有组氨酸残基被修饰,而酪氨酸残基未被修饰,修饰前后酶的氨基含量均无变化。反应动力学表明,在35℃下,GAO中有一个pK为6.5的解离基团和催化活性有关,其解离⊿H为31610 J/mol。因此组氨酸残基为GAO催化活性的一个必需基团。     

嗜水气单胞菌胞外蛋白酶的化学修饰

 蛋白酶是嗜水气单胞菌 (Aeromonashydrophila)的重要致病因子 .为研究其结构与功能之间的关系 ,用DEPC、EDC、PMSF、N AI等 9种化学修饰剂处理嗜水气单胞菌J 1株胞外蛋白酶ECPase54,然后检测残余酶活力 ,借以研究酶分子中氨基酸侧链基团与酶活性中心的关系 .结果表明 ,羧基、丝氨酸、ε 氨基、胍基等残基与酶活性无关 ;半胱氨酸残基与酶活性也无直接关系 ;而色氨酸、组氨酸、酪氨酸残基侧链以及二硫键的化学修饰引起酶活性的大幅度的下降 ,说明色氨酸、组氨酸、酪氨酸残基以及二硫键是酶活力所必需的基团     

真养产碱杆菌112R4的二羧酸单酰胺酰胺水解酶

在一株具有环酰亚胺转化活性的真养产碱杆菌112R4中发现了一种特异性的二羧酸单酰胺酰胺水解酶（半酰胺酶）,它催化环酰亚胺代谢的第二步反应,将二羧酸单酰胺水解为二羧酸和氨。该酶的底物仅限于此代谢途径的第一个酶——酰亚胺酶的产物二羧酸单酰胺,而对其它的酰胺类化合物没有明显水解活性。真养产碱杆菌112R4中的半酰胺酶和酰亚胺酶在表达上具有相关性,环酰亚胺（如琥珀酰亚胺）和二羧酸单酰胺（如琥珀酰胺酸）对它们有正调控作用,游离氨离子显示出负调控作用,琥珀酸则在酶合成和活性两方面均表现出影响作用。对重组大肠杆菌中表达的半酰胺酶粗酶的部分性质进行了研究。钴离子对半酰胺酶的活性表现出促进作用,比活力提高到3.37倍,表明半酰胺酶可能是一种金属结合酶。     

Allantoate amidinohydrolase (Allantoicase) from Chlamydomonas reinhardtii: its purification and catalytic and molecular characterization

An allantoate-degrading enzyme has been purified to electrophoretic homogeneity for the first time from a photosynthetic organism, the unicellular green algae Chlamydomonas reinhardtii. The purification procedure included a differential protein extraction followed by conventional steps such as ammonium sulfate fractionation, gel filtration, anion exchange chromatography, and preparative electrophoresis. Under the routine assay conditions (7 mM allantoate), specific activity for the purified enzyme was 185 U/mg, which rose to 225 U/mg under kinetic considerations (saturating substrate). Therefore, a turnover number of 4.5 x 10(4) min(-1) can be deduced for the 200-kDa protein. The enzyme is a true allantoicase (EC 3.5.3.4) that catalyzes the degradation of allantoate to (-)ureidoglycolate and (+)ureidoglycolate to glyoxylate. The enzyme exhibited hyperbolic kinetic for allantoate and ureidoglycolate with K(m) values of 2 and 0.7 mM, respectively. V(max) of the reaction with allantoate as substrate was nine times higher than that with ureidoglycolate. The native enzyme has a molecular weight of 200 kDa and consists of six identical or similar-sized subunits of 34 kDa each, organized in two trimers of 100 kDa. Each subunit has five cysteine residues, four of which are involved in disulfide bonds, with a total of 12 disulfide bonds in the 200-kDa protein. Allantoate inhibits competitively the reaction with ureidoglycolate as substrate. In addition, buffers and group-specific reagents affect the activity in the same manner irrespective of the substrate used. Those results suggest that both substrates use the same active site. The effect of group-specific reagents suggest that the amino acids histidine, tyrosine, and cysteine are essentials for the allantoicase activity with both substrates.     

水稻氨基酰化酶的分离纯化与性质分析

氨基酰化酶（N－acylamino－acidamidohydrolase或acylaseⅠ,EC3．5．1．14）是专一水解N-酰基化L-氨基酸的蛋白酶．从水稻黄化苗得到的抽提液,经过硫酸铵分级沉淀、丙酮分级沉淀和阴离子交换层析三个步骤,纯化得到了该酶,比活达到100U／mg蛋白,在无还原剂存在的SDS-聚丙烯酰胺凝胶电泳上显单一条带,分子量为40kD．而凝胶层析分析表明活性分子的分子量约90kD,因此可推测它的活性分子由两个亚基通过非共价键作用组合而成．进一步研究此酶的性质,在所测的五种乙酰化氨基酸中,最适底物为N-乙酰-L-甲硫氨酸．该酶的最适温度为50℃,最适pH为7．0～8．0．Co2＋和Zn2＋能增强酶活性,但烷基化试剂对酶活性没有影响,表明酶活性中心不含活化的巯基或羟基基因     

Biochemical characterisation of an allantoate-degrading enzyme from French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>): the requirement of phenylhydrazine

In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined “in vivo” using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37°C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.     

Ureide Catabolism in Soybeans : III. Ureidoglycolate Amidohydrolase and Allantoate Amidohydrolase Are Activities of an Allantoate Degrading Enzyme Complex

We demonstrate that allantoate is catabolized in soybean seedcoat extracts by an enzyme complex that has allantoate amidohydrolase and ureidoglycolate amidohydrolase activities. Soybean seedcoat extracts released ¹⁴CO₂ from [ureido-¹⁴C]ureidoglycolate under conditions in which urease is not detectable. CO₂ and glyoxylate are enzymically released in a one to one ratio indicating that ureidoglycolate amidohydrolase is the responsible activity. Ureidoglycolate amidohydrolase has a K_m of 85 micromolar for ureidoglycolate. Glyoxylate and CO₂ are enzymically released from allantoate at linear rates in a one to 2.3 ratio from 5 to 30 min. This ratio is consistent with the degradation of allantoate to two CO₂ and one glyoxylate with approximately 23% of the allantoate degraded reacting with 2-mercaptoethanol to yield 2-hydroxyethylthio, 2′-ureido, acetate (RG Winkler, JC Polacco, DG Blevins, DD Randall 1985 Plant Physiol 79: 787-793). That [¹⁴C]urea production from [2,7-¹⁴C]allantoate is not detectable indicates that allantoate-dependent glyoxylate production is enzymic and not a result of nonenzymic hydrolysis of a ureido intermediate (nonenzymic hydrolysis releases urea). These results and those from intact tissue studies (RG Winkler DG Blevins, JC Polacco, DD Randall 1987 Plant Physiol 83: 585-591) suggest that soybeans have a second amidohydrolase reaction (ureidoglycolate amidohydrolase) that follows allantoate amidohydrolase in allantoate catabolism. The rate of ¹⁴CO₂ release from [2,7-¹⁴C]allantoate is not reduced when the volume of the reaction mixture is increased, suggesting that the release of ¹⁴CO₂ is not dependent on the accumulation of free intermediates. That [2,7-¹⁴C]allantoate dependent ¹⁴CO₂ release is not proportionally diluted by unlabeled ureidoglycolate indicates that the reaction is carried out by an enzyme complex. This is the first report of ureidoglycolate amidohydrolase activity in any organism and the first in vitro demonstration in plants that the ureido-carbons of allantoate can be completely degraded to CO₂ without a urea intermediate.     

Crystal Structure of Metal-Dependent Allantoinase from Escherichia coli

Allantoinase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of (S)-allantoin into allantoate, the final step in the ureide pathway. Despite limited sequence similarity, biochemical studies of the enzyme suggested that allantoinase belongs to the amidohydrolase family. In this study, the crystal structure of allantoinase from Escherichia coli was determined at 2.1 Å resolution. The enzyme consists of a homotetramer in which each monomer contains two domains: a pseudo-triosephosphate-isomerase barrel and a β-sheet. Analogous to other enzymes in the amidohydrolase family, allantoinase retains a binuclear metal center in the active site, embedded within the barrel fold. Structural analyses demonstrated that the metal ions in the active site ligate one hydroxide and six residues that are conserved among allantoinases from other organisms. Functional analyses showed that the presence of zinc in the metal center is essential for catalysis and enantioselectivity of substrate. Both the metal center and active site residues Asn94 and Ser317 play crucial roles in dictating enzyme activity. These structural and functional features are distinctively different from those of the metal-independent allantoinase, which was very recently identified.     

Asparagine and boric Acid cause allantoate accumulation in soybean leaves by inhibiting manganese-dependent allantoate amidohydrolase

Our previous work demonstrated substantial accumulation of allantoate in leaf tissue of nodulated soybeans (Glycine max L. Merr., cv Williams) in response to nitrogen fertilization. Research was continued to determine the effect of nitrate and asparagine on ureide assimilation in soybean leaves. Stem infusion of asparagine into ureide-transporting soybeans resulted in a significant increase in allantoate concentration in leaf tissue. Accumulation of allantoate was also observed when asparagine was supplied in the presence of allopurinol, an inhibitor of xanthine dehydrogenase in the pathway of ureide biosynthesis. In vitro, asparagine was found to have an inhibitory effect on the activity of allantoate amidohydrolase, a Mn²⁺-dependent enzyme catalyzing allantoate breakdown in soybean leaves. The inhibition was partially overcome by supplemental Mn²⁺ in enzyme assays. Another inhibitor of allantoate amidohydrolase, boric acid, applied foliarly on field-grown nodulated soybeans, caused up to a 10-fold increase in allantoate content of leaf tissue. Accumulation of allantoate in response to boric acid was either eliminated or greatly reduced in plants presprayed with Mn²⁺. We conclude that elevated levels of allantoate in leaves of ureide-transporting soybeans fertilized with ammonium nitrate result from inhibition of allantoate degradation by asparagine and that Mn²⁺ is a critical factor in this inhibition. Furthermore, our studies with asparagine and boric acid indicate that availability of Mn²⁺ has a direct effect on ureide catabolism in soybean.     

Acetylpolyamine amidohydrolase from Mycoplana ramosa: gene cloning and characterization of the metal-substituted enzyme.

We have cloned a gene (aphA) encoding acetylpolyamine amidohydrolase from Mycoplana ramosa ATCC 49678, (previously named Mycoplana bullata). A genomic library of M. ramosa was screened with an oligonucleotide probe designed from a N-terminal amino acid sequence of the enzyme purified from M. ramosa. Nucleotide sequence analysis revealed an open reading frame of 1,023 bp which encodes a polypeptide with a molecular mass of 36,337 Da. This is the first report of the structure of acetylpolyamine amidohydrolase. The aphA gene was subcloned under the control of the trc promoter and was expressed in Escherichia coli MM294. The recombinant enzyme was purified, and the enzymatic properties were characterized. Substrate specificities, Km values, and Vmax values were identical to those of the native enzyme purified from M. ramosa. In the analysis of the metal-substituted enzymes, we found that the acid limb of pH rate profiles shifts from 7.2 for the original zinc enzyme to 6.6 for the cobalt enzyme. This change suggests that the zinc atom is essential for the catalytic activity of the enzyme similarly to the zinc atom in carboxypeptidase A.     

Soybean cultivars 'Williams 82' and 'Maple Arrow' produce both urea and ammonia during ureide degradation

The ability of two soybean (Glycine max L. [Merrill]) cultivars, 'Williams 82' and 'Maple Arrow', which were reported to use different ureide degradation pathways, to degrade the ureides allantoin and allantoate was investigated. Protein fractions and total leaf homogenates from the fourth trifoliate leaves of both cultivars were examined for the ability to evolve either (14)CO(2) or [(14)C]urea from (14)C-labelled ureides in the presence of various inhibitors. (14)CO(2) evolution from [2,7-(14)C]allantoate was catalysed by 25-50% saturated ammonium sulphate fractions of both cultivars. This activity was inhibited by acetohydroxamate (AHA), which has been used to inhibit plant ureases, but not by phenylphosphorodiamidate (PPD), a more specific urease inhibitor. Thus, in both cultivars, allantoate may be metabolized by allantoate amidohydrolase. This activity was sensitive to EDTA, consistent with previous reports demonstrating that allantoate amidohydrolase requires manganese for full activity. Total leaf homogenates of both cultivars evolved both (14)CO(2) and [(14)C]urea from [2,7-(14)C] (ureido carbon labelled) allantoin, not previously reported in either 'Williams 82' or in 'Maple Arrow'. In situ leaf degradation of (14)C-labelled allantoin confirmed that both urea and CO(2)/NH(3) are direct products of ureide degradation. Growth of plants in the presence of PPD under fixing and non-fixing conditions caused urea accumulation in both cultivars, but did not have a significant impact on total seed nitrogen. Urea levels were higher in N-fixing plants of both cultivars. Contrary to previous reports, no significant biochemical difference was found in the ability of these two cultivars to degrade ureides under the conditions used.     

Anandamide amidohydrolase (fatty acid amide hydrolase)

Anandamide (N-arachidonoylethanolamine) loses its cannabimimetic activity when it is hydrolyzed to arachidonic acid and ethanolamine by the catalysis of an enzyme referred to as anandamide amidohydrolase or fatty acid amide hydrolase. Cravatt's group and our group cloned cDNA of the enzyme from rat, human, mouse and pig, and the primary structures revealed that the enzymes belong to an amidase family characterized by the amidase signature sequence. The recombinant enzyme acted not only as an amidase for anandamide and oleamide, but also as an esterase for 2-arachidonoylglycerol. The reversibility of the enzymatic anandamide hydrolysis and synthesis was also confirmed with a purified recombinant enzyme. Several fatty acid derivatives like methyl arachidonyl fluorophosphonate potently inhibited the enzyme. The enzyme was distributed widely in mammalian organs such as liver, small intestine and brain. However, the anandamide hydrolyzing enzyme found in human megakaryoblastic cells was catalytically distinct from the previously known enzyme.