Metabolism of 4-chloro-2-methylphenoxyacetate by a soil pseudomonad. Preliminary evidence for the metabolic pathway

1. A pseudomonad capable of utilizing the herbicide 4-chloro-2-methylphenoxyacetate as a sole carbon source was isolated from soil and cultured in liquid medium. 2. Analysis of induction patterns of 4-chloro-2-methylphenoxyacetate-grown cells suggests that 5-chloro-o-cresol and 5-chloro-3-methylcatechol are early intermediates in the oxidation of 4-chloro-2-methylphenoxyacetate. Cells were not adapted to oxidize 4-chloro-6-hydroxy-2-methylphenoxyacetate. 3. In culture, 4-chloro-2-methylphenoxyacetate rapidly disappeared and the chlorine in the molecule was quantitatively released as Cl(-) ion. 4. A lactone (gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide) was isolated from cultures and established as an intermediate. 5. The following metabolic pathway is suggested: 4-chloro-2-methylphenoxyacetate --> 5-chloro-o-cresol --> 5-chloro-3-methylcatechol --> cis-cis-gamma-chloro-alpha-methylmuconate --> gamma-carboxymethylene-alpha-methyl-Delta(alphabeta)-butenolide --> gamma-hydroxy-alpha-methylmuconate. 6. The tentative identification of 5-chloro-o-cresol, a gamma-chloro-alpha-methylmuconate and gamma-hydroxy-alpha-methylmuconate in culture extracts supports this scheme. However, the catechol was never observed to accumulate in cultures. 7. The detection of 4-chloro-6-hydroxy-2-methylphenoxyacetate, 2-methyl-phenoxyacetate, a dehalogenated cresol and oxalate in culture extracts is discussed in relation to the proposed metabolic pathway.     

Bacterial metabolism of 4-chlorophenoxyacetate

1. A pseudomonad capable of utilizing 4-chlorophenoxyacetate (CPA) as sole source of organic carbon was isolated from soil. 2. The organism was grown in liquid culture and the following compounds were isolated and identified in culture extracts: 4-chloro-2-hydroxyphenoxyacetate, 4-chlorocatechol, beta-chloromuconate probably the cis-trans isomer and gamma-carboxymethylene-Delta(alphabeta)-butenolide. 3. Cells grown on 4-chlorophenoxyacetate were able to metabolize 4-chloro-2-hydroxyphenoxyacetate, 4-chlorocatechol and gamma-carboxymethylene-Delta(alphabeta)-butenolide without a lag period. They were not adapted to 4-chlorophenol, or to either culture isolated or synthetic beta-chloromuconate, possibly because of stereospecificity towards the cis-cis isomer. 4. On the basis of isolation and induction evidence, the following metabolic pathway is proposed for the breakdown of 4-chlorophenoxyacetate by this organism: 4-chlorophenoxyacetate --> 4-chloro-2-hydroxyphenoxyacetate --> 4-chlorocatechol --> cis-cis-beta-chloromuconate --> gamma-carboxymethylene-Delta(alphabeta)-butenolide --> maleylacetate and fumarylacetate --> fumarate and acetate.     

Cuprous oxidase activity of yeast Fet3p and human ceruloplasmin: implication for function

The Fet3 protein in Saccharomyces cerevisiae and mammalian ceruloplasmin are multicopper oxidases (MCO) that are required for iron homeostasis via their catalysis of the ferroxidase reaction, 4Fe(2+)+O(2)+4H(+)-->4Fe(3+)+2H(2)O. The enzymes may play an essential role in copper homeostasis since they exhibit a strikingly similar kinetic activity towards Cu(1+) as substrate. In contrast, laccase, an MCO that exhibits weak activity towards Fe(2+), exhibits a similarly weak activity towards Cu(1+). Kinetic analyses of the Fet3p reaction demonstrate that the ferroxidase and cuprous oxidase activities are due to the same electron transfer site on the enzyme. These two ferroxidases are fully competent kinetically to play a major role in maintaining the cuprous-cupric redox balance in aerobic organisms.     

Bacterial metabolism of 2,4-dichlorophenoxyacetate

1. Two Pseudomonas strains isolated from soil metabolized 2,4-dichlorophenoxyacetate (2,4-D) as sole carbon source in mineral salts liquid medium. 2. 2,4-Dichlorophenoxyacetate cultures of Pseudomonas I (Smith, 1954) contained 2,4-dichlorophenol, 2-chlorophenol, 3,5-dichlorocatechol and alpha-chloromuconate, the last as a major metabolite. 3. Dechlorination at the 4(p)-position of the aromatic ring must therefore take place at some stages before ring fission. 4. Pseudomonas N.C.I.B. 9340 (Gaunt, 1962) cultures metabolizing 2,4-dichlorophenoxyacetate contained 2,4-dichloro-6-hydroxyphenoxyacetate, 2,4-dichlorophenol, 3,5-dichlorocatechol and an unstable compound, probably alphagamma-dichloromuconate. 5. Cell-free extracts of the latter organism grown in 2,4-dichlorophenoxyacetate cultures contained an oxygenase that converted 3,5-dichlorocatechol into alphagamma-dichloromuconate, a chlorolactonase that in the presence of Mn(2+) ions converted the dichloromuconate into gamma-carboxymethylene-alpha-chloro-Delta(alphabeta)-butenolide, and a delactonizing enzyme that gave alpha-chloromaleylacetate from this lactone. 6. Pathways of metabolism of 2,4-dichlorophenoxyacetate are discussed.     

Metal cofactor requirement of β-lactamase II

1. The apoenzyme obtained on removal of Zn(2+) from beta-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn(2+)-containing enzyme. 2. Removal of Zn(2+) led to a conformational change in the enzyme and partial unmasking of a thiol group. 3. Replacement of Zn(2+) by Co(2+), Cd(2+), Mn(2+) or Hg(2+) gave enzymes with significant, but lower, beta-lactamase activity. No activity was detected in the presence of Cu(2+), Ni(2+), Mg(2+) or Ca(2+). 4. Equilibrium dialysis indicated that the enzyme had at least two Zn(2+) binding sites. With benzylpenicillin as substrate the variation in activity with concentration of Zn(2+) indicated that activity paralleled binding of Zn(2+) to the site of highest affinity. 5. Replacement of Zn(2+) by Co(2+) and Cd(2+) gave enzymes with absorption bands at 340 and 245nm respectively, and raised the question of whether the thiol group in the enzyme is a metal-ion ligand. 6. Reduction of the product obtained by reaction of denatured beta-lactamase II with Ellman's reagent [5,5'-dithiobis-(2-nitrobenzoic acid)] gave a protein which could refold to produce beta-lactamase II activity in high yield.     

The nature of the collagenolytic cathepsin of rat liver and its distribution in other rat tissues

1. An enzyme present in rat liver extracts degraded insoluble collagen maximally at pH3.5. Collagenolytic activity was more abundant in kidney, spleen and bone marrow and was also present in decreasing concentrations in ileum, lung, heart, skin and muscle. 2. The crude collagenolytic cathepsin was activated by cysteine and dithiothreitol, but not by 2-mercaptoethanol. Iodoacetamide, p-chloromercuribenzoate and 7-amino-1-chloro-3-l-tosylamidoheptan-2-one hydrochloride inhibited the enzyme. Zn(2+), Fe(3+) and Hg(2+) ions were strongly inhibitory, but Ca(2+), Co(2+), Mg(2+) and Fe(2+) ions had little or no effect. EDTA was an activator of the enzyme. Inhibitors of cathepsin B were found to enhance collagenolysis, but phenylpyruvic acid, a cathepsin D inhibitor, inhibited the enzyme. Di-isopropyl phosphorofluoridate had no effect. 3. Collagenolysis at pH3.5 and 28 degrees C was restricted to cleavage of the telopeptide region in insoluble collagen, and the material that was solubilized consisted mostly of alpha-chains. 4. The collagenolytic cathepsin was separated from cathepsins B2 and D by fractionation on Sephadex G-100 and a partial separation from cathepsin B1 was obtained by chromatography on DEAE-Sephadex. 5. The function of the collagenolytic cathepsin in the catabolism of collagen is discussed in relation to the action of the other lysosomal proteinases and the neutral collagenase.     

Effects of some metal ions on human erythrocyte glutathione reductase: an in vitro study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.     

A metal bridge between two enzyme families. 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Aquifex aeolicus requires a divalent metal for activity

The enzymes 3-deoxy-d-manno-octulosonic acid-8-phosphate synthase (KDO8PS) and 3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase (DAHPS) catalyze analogous condensation reactions between phosphoenolpyruvate and d-arabinose 5-phosphate or d-erythrose 4-phosphate, respectively. While several similarities exist between the two enzymatic reactions, classic studies on the Escherichia coli enzymes have established that DAHPS is a metalloenzyme, whereas KDO8PS has no metal requirement. Here, we demonstrate that KDO8PS from Aquifex aeolicus, representing only the second member of the KDO8PS family to be characterized in detail, is a metalloenzyme. The recombinant KDO8PS, as isolated, displays an absorption band at 505 nm and contains approximately 0.4 and 0.2-0.3 eq of zinc and iron, respectively, per enzyme subunit. EDTA inactivates the enzyme in a time- and concentration-dependent manner and eliminates the absorption at 505 nm. The addition of Cu(2+) to KDO8PS produces an intense absorption at 375 nm, while neither Co(2+) nor Ni(2+) produce such an effect. The EDTA-treated enzyme is reactivated by a wide range of divalent metal ions including Ca(2+), Cd(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+) and is reversibly inhibited by higher concentrations (>1 mm) of certain metals. Analysis of several metal forms of the enzyme by plasma mass spectrometry suggests that the enzyme preferentially binds one, two, or four metal ions per tetramer. These observations strongly suggest that A. aeolicus KDO8PS is a metalloenzyme in vivo and point to a previously unrecognized relationship between the KDO8PS and DAHPS families.     

Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.     

Linoleic acid isomerase in Lactobacillus plantarum AKU1009a proved to be a multi-component enzyme system requiring oxidoreduction cofactors

Linoleic acid isomerase in Lactobacillus plantarum was found to be a novel multi-component enzyme system widespread in membrane and soluble fractions. The isomerization reaction involved a hydration step, 10-hydroxy-12-octadecenoic acid production from linoleic acid, as part of the reaction, and the hydration reaction was catalyzed by the membrane fraction. Both membrane and soluble fractions were required for the whole isomerization reaction, i.e., conjugated linoleic acid (CLA) production from linoleic acid, and for CLA production from 10-hydroxy-12-octadecenoic acid, a reaction intermediate. The multi-component enzyme system was inhibited by o-phenanthroline, and divalent metal ions such as Ni(2+) and Co(2+) restored activity. Metal oxides such as VO(4)(3+), MoO(4)(2+), and MnO(4)(2+) enhanced activity. The multi-component enzyme systems required oxidoreduction cofactors such as NADH together with FAD or NADPH for total activity.     

Studies on a testosterone glucuronyltransferase from the cytosol fraction of human liver

An enzyme that conjugates the 16alpha-hydroxyl group of oestriol with glucuronic acid was found in the cytosol fraction of human liver. The enzymic activity could not be sedimented when the cytosol fraction was centrifuged at 158000g(av.) for 120min. The oestriol 16alpha-glucuronyltransferase was purified 100-fold by 0-30% saturation of the cytosol fraction with ammonium sulphate followed by filtration of the precipitate through Sephadex G-200. The activity was eluted at the void volume. The product of the reaction, oestriol 16alpha-monoglucuronide, was identified by paper chromatography and by crystallization of radioactive product to constant specific radioactivity. The optimum temperature was 37 degrees C, and the activation energy was calculated to be 11.1kcal/mol. The apparent Michaelis-Menten constants for oestriol and UDP-glucuronic acid were 13.3 and 100mum respectively. Cu(2+), Zn(2+) and Hg(2+) inhibited, whereas Mg(2+), Mn(2+) and Fe(2+) stimulated the enzyme. Substrate-specificity studies indicated that the amount of oestradiol-17beta, oestradiol-17alpha and oestrone conjugated was not more than about 5% of that found for oestriol. Oestriol 16alpha-monoglucuronide, a product of the reaction, did not inhibit the 16alpha-oestriol glucuronyltransferase; in contrast, UDP, another product of the reaction, inhibited the enzyme competitively with respect to UDP-glucuronic acid as the substrate, and non-competitively with respect to oestriol as the substrate. ATP and UDP-N-acetylglucosamine did not affect the oestriol 16alpha-glucuronyltransferase. 17-Epioestriol acted as a competitive inhibitor and 16-epioestriol as a non-competitive inhibitor of the glucuronidation of oestriol. 5alpha-Pregnane-3alpha,20alpha-diol also inhibited the enzyme non-competitively. It is most likely that the oestriol 16alpha-glucuronyltransferase described here is bound to the membranes of the endoplasmic reticulum.     

Coproporphyrinogenase in tobacco (Nicotiana tabacum L.)

1. Coproporphyrinogenase was extracted and purified from tobacco (Nicotiana tabacum L.). Enzyme activity was mainly located in mitochondria rather than in chloroplasts. The enzyme was purified by differential centrifugation, ammonium sulphate fractionation, calcium phosphate gel adsorption and dialysis. A 69-fold final purification was obtained. 2. An apparent K(m) value of 3.6x10(-5)m was found, the value being largely dependent on the amount of coproporphyrin III recovered after reduction with sodium amalgam to coproporphyrinogen III. Protoporphyrin formation was linear up to 3h and decreased with further incubation. The enzyme activity increased with the concentration of enzyme protein up to 30mug/ml of solution. 3. Enzyme activity was greatly enhanced by increasing Fe(2+) concentrations up to 0.5mm, beyond which inhibition occurred. Co(2+) and Mn(2+) were also found to activate at low concentrations (0.1mm) and inhibit at higher concentrations (5mm). Fe(3+) and Cu(2+), both at 0.1mm, and o-phenanthroline and EDTA, each at 1mm, were found to be inhibitory.     

Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).     

Bacterial metabolism of 4-chloro-2-methylphenoxyacetate. Formation of glyoxylate by side-chain cleavage

Crude extracts of Pseudomonas sp. grown on 4-chloro-2-methylphenoxyacetate as sole source of carbon were shown to oxidize 4-chloro-2-methylphenoxyacetate to 5-chloro-o-cresol and glyoxylate. A labelled 2,4-dinitrophenylhydrazone was isolated from an incubation mixture in which 4-chloro-2-methylphenoxy[carboxy-¹⁴C]acetate was used. The hydrazone was shown to behave identically on thin-layer chromatograms with the authentic 2,4-dinitrophenylhydrazone of glyoxylate. Radioactivity assay showed that 82% of the side chain of 4-chloro-2-methylphenoxyacetate was recovered as glyoxylate.     

Iron-induced oxidative damage of corn root plasma membrane H(+)-ATPase

The effect of iron on the activity of the plasma membrane H(+)-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated. In the presence of either Fe(2+) or Fe(3+) (100-200 microM of FeSO(4) or FeCl(3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed. Half-maximal inhibition was attained at 25 microM and 50 microM for Fe(2+) and Fe(3+), respectively. Inhibition of the ATPase activity was prevented in the presence of metal ion chelators such as EDTA, deferoxamine or o-phenanthroline in the incubation medium. However, preincubation of CRMF in the presence of 100 microM Fe(2+), but not with 100 microM Fe(3+), rendered the ATPase activity (measured in the presence of excess EDTA) irreversibly inhibited. Inhibition was also observed using a preparation further enriched in plasma membranes by gradient centrifugation. Addition of 0.5 mM ATP to the preincubation medium, either in the presence or in the absence of 5 mM MgCl(2), reduced the extent of irreversible inhibition of the H(+)-ATPase. Addition of 40 microM butylated hydroxytoluene and/or 5 mM dithiothreitol, or deoxygenation of the incubation medium by bubbling a stream of argon in the solution, also caused significant protection of the ATPase activity against irreversible inhibition by iron. Western blots of CRMF probed with a polyclonal antiserum against the yeast plasma membrane H(+)-ATPase showed a 100 kDa cross-reactive band, which disappeared in samples previously exposed to 500 microM Fe(2+). Interestingly, preservation of the 100 kDa band was observed when CRMF were exposed to Fe(2+) in the presence of either 5 mM dithiothreitol or 40 microM butylated hydroxytoluene. These results indicate that iron causes irreversible inhibition of the corn root plasma membrane H(+)-ATPase by oxidation of sulfhydryl groups of the enzyme following lipid peroxidation.     

Mechanism of non-enzymic transamination reaction between histidine and α-oxoglutaric acid

Non-enzymic transamination reactions at 85 degrees between various amino acids and alpha-oxoglutaric acid are catalysed by metal ions, e.g. Al(3+), Fe(2+), Cu(2+) and Fe(3+). The reaction is optimum at pH4.0. Of the 14 amino acids studied histidine is the most active. In the presence of Al(3+) histidine transaminates with alpha-oxoglutaric acid, forming glutamic acid and Al(3+)-imidazolylpyruvic acid complex as the end products. However, in the presence of Fe(2+) or Cu(2+) the products are glutamic acid and a 1:2 metal ion-imidazolylpyruvic acid chelate. The greater effectiveness of histidine in these reactions is attributed to the presence of the tertiary imidazole nitrogen atom, which is involved in the formation of stable sparingly soluble metal ion-imidazolylpyruvic acid complexes or chelates as end products of these reactions. Of the metal ions studied only Al(3+), Fe(2+), Fe(3+) and Cu(2+) are effective catalysts for the transamination reactions, and EDTA addition completely inhibits the catalytic effect of the Al(3+). Spectrophotometric evidence is presented to demonstrate the presence of metal ion complexes of Schiff bases of histidine as intermediates in the transamination reactions. These results may contribute to understanding the role of histidine in enzyme catalysis.     

Slow binding of metal ions to pigeon liver malic enzyme: a general case

Pigeon liver malic enzyme was inhibited by lutetium ion through a slow-binding process, which resulted in a concave down tracing of the enzyme activity assay. The fast initial rates were independent of lutetium ion concentration, while the slow steady-state rates decreased with increasing Lu(3+) concentration. The observed rate constant for the transition from initial rate to steady-state rate, k(obs), exhibited saturation kinetics as a function of Lu(3+) concentration, suggesting the involvement of an isomerization process between two enzyme forms (R-form and T-form). The binding affinity of Lu(3+) to the R-form is weaker (K(d,Lu) = 14 microM) than that of Mn(2+) (K(m,Mn) = 1.89 microM); however, Lu(3+) has much tighter binding affinity with the T-form ( = 0.83 microM). Lu(3+) was shown to be a competitive inhibitor with respect to Mn(2+), which suggests that Lu(3+) and Mn(2+) are competing for the same metal binding site of the enzyme. These observations are in accordance with the available crystal structure information, which shows a distorted active site region of the Lu(3+)-containing enzyme. Other divalent cations, i.e., Fe(2+), Cu(2+), or Zn(2+), also act as time-dependent slow inhibitors for malic enzyme. The dynamic quenching constants of the intrinsic fluorescence for the metal-free and Lu(3+)-containing enzymes are quite different, indicating the conformational differences between the two enzyme forms. The secondary structure of these two enzyme forms, on the other hand, was not changed. The above results indicated that replacement of the catalytically essential Mn(2+) by other metal ions leads to a slow conformational change of the enzyme and consequently alters the geometry of the active site. The transformed enzyme conformation, however, is unfavorable for catalysis. Both the chemical nature of the metal ion and its correct coordination in the active site are essential for catalysis.     

Purification of the alliin lyase of garlic, Allium sativum L

1. Alliin lyase (EC 4.4.1.4) was purified up to sevenfold from garlic-bulb homogenates. The enzyme was unstable to storage at -10 degrees , particularly in dilute concentrations, but the addition of glycerol (final concentration 10%, v/v) stabilized the activity completely for at least 30 days. 2. The purified enzyme had an optimum pH for activity at 6.5. The addition of pyridoxal phosphate stimulated the reaction rate and the stimulation became more marked as the purification proceeded. 3. Hydroxylamine (10mum) and cysteine (0.5mm) inhibited the enzyme activity by more than 80%. Spectral studies indicated that cysteine reacted with pyridoxal phosphate bound to the protein. 4. The K(m) values for S-methyl-, S-ethyl-, S-propyl-, S-butyl- and S-allyl-l-cysteine sulphoxides were determined. With S-allyl-l-cysteine sulphoxide the K(m) was 6mm and the V(max.) was greater than those with the other substrates tested. 5. The thioether analogues of the substrates were competitive inhibitors for the lyase reaction. The K(i) decreased with increasing chain length of the alkyl substituent. With S-ethyl-l-cysteine sulphoxide as substrate the K(i) was 33, 8 and 5mm respectively for S-methyl-, S-ethyl- and S-propyl-l-cysteine. 6. The addition of EDTA or Mg(2+), Mn(2+), Co(2+) or Fe(2+) stimulated the reaction rate. Other bivalent cations either had no effect or gave a strong inhibition. In the presence of EDTA no further increase of activity was observed with added Mg(2+).     

虎纹捕鸟蛛毒液中透明质酸酶的纯化和部分性质的研究

用分子筛（岛津ＤＩＯＬ－１５０柱）和阳离子交换（岛津ＷＣＸ－１柱）高效液相色谱从虎纹捕鸟蛛（Ｓｅｌｅｎｏｃｏｓｍｉａｈｕｗｅｎａ）毒液中分离提纯透明质酸酶（Ｈｙａｌｕｒｏｎｉｄａｓｅ,ＥＣ３．２．１．３５）．经等电聚焦电泳为一条带,ｐＩ＝７．２．经ＳＤＳ－聚丙烯酰胺凝胶电泳测得分子量为４０ｋＤ,经凝胶过滤测得分子量为４０．７ｋＤ。以透明质酸为底物时在ｐＨ３．５─５．５范围内有较大活性,最适ｐＨ值为４．０;在ｐＨ４．５─６．０范围内稳定,在反应温度为３０─６０℃时有较大活性,最适温度为５０℃;对热稳定,０．１５ｍｏｌ／Ｌ的ＮａＣｌ对酶活性有一定的稳定作用．３％的肝素、５００μｍｏｌ／Ｌ的Ｈｇ~（２＋）、Ｆｅ~（２＋）、Ｃｕ~（２＋）对酶活性有明显的抑制作用。     

Mutations in the ribonuclease H active site of HIV-RT reveal a role for this site in stabilizing enzyme-primer-template binding

The RNase H activity of HIV-RT is coordinated by a catalytic triad (E478, D443, D498) of acidic residues that bind divalent cations. We examined the effect of RNase H deficient E(478)-->Q and D(549)-->N mutations that do not alter polymerase activity on binding of enzyme to various nucleic acid substrates. Binding of the mutant and wild-type enzymes to various nucleic acid substrates was examined by determining dissociation rate constants (k(off)) by titrating both Mg(2+) and salt concentrations. In agreement with the unaltered polymerase activity of the mutant, the k(off) values for the wild-type and mutant enzymes were essentially identical using DNA-DNA templates in the presence of 6 mM Mg(2+). However, with lower concentrations of Mg(2+) and in the absence of Mg(2+), although both enzymes dissociated more rapidly, the mutant enzymes dissociated several-fold more slowly than the wild type. This was also observed on RNA-DNA templates. These results indicate that alterations in residues essential for Mg(2+) binding have a pronounced positive effect on enzyme-template stability and that the negative residues in the RNase H region of the enzyme have a negative influence on binding in the absence of Mg(2+). In this regard RT is similar to other nucleic acid cleaving enzymes that show enhanced binding upon mutation of active site residues.