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1.
Recently, the synthesis and properties of several 6-substituted flavins as active site probes for flavoproteins have been reported (Ghisla, S., Massey, V., and Yagi, K. (1986) Biochemistry 25, 3282-3289). Here, we report results of experiments in which 6-thiocyanato-FAD and 6-mercapto-FAD have been substituted for the native flavin of phenol hydroxylase. The 6-SCN-FAD enzyme was converted spontaneously to the 6-mercaptoflavin form probably due to dissociation of flavin, followed by attack of external protein thiols. The pK alpha values of uncomplexed and phenol-bound 6-mercapto-FAD enzyme were determined. Both the spontaneously formed 6-mercapto-FAD enzyme and the enzyme reconstituted with preformed 6-mercapto-FAD were treated with a variety of thiol-specific reagents, and reaction rates were followed by spectroscopic means. Comparison with the corresponding rates found with free flavin suggested a high degree of accessibility to the flavin 6-position. Accessibility was somewhat decreased in the presence of phenol. Upon treatment with low concentrations of methyl methanethiosulfonate or N-ethylmaleimide (NEM), extremely rapid spectral changes were apparent. The former reaction, however, was reversed spontaneously within 2 h. Reaction with NEM was biphasic, with spectral changes consistent with the mechanism previously proposed (Steenkamp, D. J., McIntire, W., and Kenney, W. C. (1978) J. Biol. Chem. 253, 2818-2824), followed by a small absorbance decrease due to protein conformational changes. The NEM reaction is unusual, being easily reversed by addition of excess dithiothreitol. 相似文献
2.
3.
Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway. 相似文献
4.
Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2. 总被引:8,自引:7,他引:1
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Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway. 相似文献
5.
Purification and characterization of a bacterial nitrophenol oxygenase which converts ortho-nitrophenol to catechol and nitrite. 总被引:10,自引:4,他引:6
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A nitrophenol oxygenase which stoichiometrically converted ortho-nitrophenol (ONP) to catechol and nitrite was isolated from Pseudomonas putida B2 and purified. The substrate specificity of the enzyme was broad and included several halogen- and alkyl-substituted ONPs. The oxygenase consisted of a single polypeptide chain with a molecular weight of 58,000 (determined by gel filtration) or 65,000 (determined on a sodium dodecyl sulfate-polyacrylamide gel). The enzymatic reaction was NADPH dependent, and one molecule of oxygen was consumed per molecule of ONP converted. Enzymatic activity was stimulated by magnesium or manganese ions, whereas the addition of flavin adenine dinucleotide, flavin mononucleotide, or reducing agents had no effect. The apparent Kms for ONP and NADPH were 8 and 140 microM, respectively. 2,4-Dinitrophenol competitively (Ki = 0.5 microM) inhibited ONP turnover. The optimal pH for enzyme stability and activity was in the range of 7.5 to 8.0. At 40 degrees C, the enzyme was totally inactivated within 2 min; however, in the presence of 1 mM ONP, 40% of the activity was recovered, even after 10 min. Enzymatic activity was best preserved at -20 degrees C in the presence of 50% glycerol. 相似文献
6.
Picolinic acids have been synthesized previously from catechols by the action of catechol 2,3-dioxygenase and a subsequent
chemical reaction in the presence of ammonia. 2-Aminophenol 1,6-dioxygenase catalyzes ring cleavage of several ortho-aminophenols. The ring fission products spontaneously convert to picolinic acids. Resting cells of Escherichia coli DH5α/pNBZ14 harboring the genes for 2-aminophenol 1,6-dioxygenase converted 2-aminophenol and 6-amino-m-cresol to picolinic acid and 5-methylpicolinic acid with yields greater than 90%. The results provide a convenient strategy
for the synthesis of substituted picolinic acids from the corresponding aminophenols. Journal of Industrial Microbiology & Biotechnology (2000) 25, 25–28.
Received 25 October 1999/ Accepted in revised form 19 April 2000 相似文献
7.
A. V. Maksimenko M. L. Petrova E. G. Tishchenko Yu. V. Shchechilina 《Russian Journal of Bioorganic Chemistry》2000,26(5):322-326
The modification of hyaluronidase by aldehydodextran regulates inhibition of the enzyme by heparin. A 70–90% modification
of the surface amino groups of hyaluronidase results in sharp conformational changes and a substantial decrease of its inhibition
by heparin, whereas hyaluronidase derivatives with a modification degree of 96–100% are practically uninhibited. 相似文献
8.
S Hari Krishna S G Prapulla N G Karanth 《Journal of industrial microbiology & biotechnology》2000,25(3):147-154
Isoamyl butyrate, an important fruity flavor ester, was synthesized using Rhizomucor miehei lipase immobilized on a weak anion exchange resin (Lipozyme IM-20) by the esterification of isoamyl alcohol and butyric acid.
The effects of various reaction parameters such as substrate and enzyme concentrations, substrate molar ratio, temperature
and incubation time have been investigated. Yields above 90% were obtained with substrate concentrations as high as 2.0 M.
No evidence of enzyme inhibition by butyric acid was present up to 1.0 M concentration. Acid inhibition and, to a small extent,
alcohol inhibition were evident above 1.0 M substrate concentration. Conversions reached a saturation value by the end of
24–48 h of incubation due to the accumulation of the water of reaction. The equilibrium was successfully pushed forward towards
esterification by removing the accumulated water using a molecular sieve.Journal of Industrial Microbiology & Biotechnology (2000) 25, 147–154.
Received 09 February 2000/ Accepted in revised form 24 June 2000 相似文献
9.
Savrasova EA Kivero AD Shakulov RS Stoynova NV 《Journal of industrial microbiology & biotechnology》2011,38(9):1287-1294
Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically
important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA)
biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol
74:2259–2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols
as new-generation biofuels (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651–657,
2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775, 2008;
Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009). On the basis of
these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol,
we also—as has been demonstrated previously (Atsumi et al. in Nature 451:86–90, 2008; Atsumi et al. in Appl Microbiol Biotechnol
85:651–657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89–98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769–5775,
2008; Shen and Liao in Metab Eng 10:312–320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471–479, 2009)—used enzymes
of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto
acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol
instead of valine. Expression of BCKAD alone also resulted in isobutanol accumulation in the culture broth, supporting earlier
obtained data (Atsumi et al. in Appl Microbiol Biotechnol 85:651–657, 2010) that native ADHs of E. coli are also capable of isobutanol production. Thus, in this work, isobutanol synthesis by E. coli was achieved using enzymes similar to but somewhat different from those previously used. 相似文献
10.
When wheat seedlings were grown in the presence of 62.5-500μM 4 chloro-5-dimethylamino-2-phenyl-3(2H) pyridazinone, an inhibitor of photosystem II electron transport, there was a marked
inhibition of in vivo photosystem II electron transport as revealed by the analysis of fast chlorophyll a fluorescence transients
in intact leaves and by the inhibition (95% at 500μM) of net photosynthesis in intact leaves Accompanying this inhibition of photosystem II electron transport, there was a decrease
in the content of photosynthetic pigments. The extent of lipid peroxidation, measured in terms of malondialdehyde content
was not increased; rather it was found decreased. An analysis of in vitro lipid peroxidation of the thylakoid membranes of
control and 4-chloro-5-dimethylamino-2-phenyl-3(2H) pyridazinone treated plants in the presence of a sensitizer dye (toluidine
blue) showed a similar rate both in the control and treated samples suggesting that the availability of unsaturated fatty
acids as a substrate for lipid peroxidation was not limiting even though it decreased in the treated plants. On the other
hand, it appears that the availability of the free radicals for lipid peroxidation was decreased byenhanced activity of the
enzyme systems involved in the metabolism of free radicals. Measurements of the activities of enzymes involved in the metabolism
of free radicals showed an increase in the activities of NADPH-glutathione reductase (6–8 fold) and catalase (15–30%) and
a decrease in the activity of superoxide dismutase (30–45%) in the treated plants. 相似文献
11.
Increased levels of rhizospheric dissolved inorganic carbon have repeatedly been demonstrated to enhance plant growth by up
to 80%, although carbon from dark fixation accounts for only 1–3% of total plant carbon gain. This study, therefore, aimed
at investigating the effects of bicarbonate on nitrate uptake, assimilation and translocation to shoots. Clonal saplings of
poplar (Populus canescens(Ait.) Sm.) and elder (Sambucus nigraL.) were grown hydroponically for 35 days in a nutrient solution containing 0, 0.5 and 1 mM bicarbonate and 2 mM nitrate as the sole nitrogen source at pH 7.0. Net nitrate uptake, root nitrate accumulation and reduction, and export of
nitrogenous solutes to shoots were measured after incubating plants with 15N-labelled nitrate for 24 h. Net nitrate uptake increased non-significantly in plant species (19–61% compared to control plants)
in response to 1 mM bicarbonate. Root nitrate reduction and nitrogen export to shoots increased by 80 and 95% and 15 and 44% in poplar and elder,
respectively. With enhanced root zone bicarbonate, both species also exhibited a marked shift between the main nitrate utilising
processes. Poplar plants increasingly utilised nitrate via nitrate reduction (73–88% of net nitrate uptake), whereas the proportions
of export (20–9%) and storage in roots (7–3%) declined as plants were exposed to 1 mM external bicarbonate. On the other hand, elder plants exhibited a significant increase of root nitrate reduction (44–66%)
and root nitrate accumulation (6–25%). Nitrate translocation to elder shoots decreased from 50 to 8% of net nitrate uptake.
The improved supply of nitrogen to shoots did not translate into a significant stimulation of growth, relative growth rates
increased by only 16% in poplar saplings and by 7% in elder plants.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
12.
Sakr Soulaimane; Gaillard Cecile; Roblin Gabriel; Delrot Serge 《Journal of experimental botany》1994,45(8):1091-1096
To understand contradictory data published in the literature,the sensitivity of sucrose and of valine uptake to N-ethylmaleimide(NEM) was reinvestigated in detail with plasma membrane vesiclespurified by phase partitioning from mature sugar beet (Betavulgaris) leaves. Uptake in the vesicles was energized by anartificial proton-motive force combining a pH gradient and anelectrical gradient. Three main parameters were varied in theexperiments: the presence of a reducing agent, dlthiothreitol(DTT) In the medium used to store the vesicles, the temperatureof pretreatment with NEM (12 or 23°C) and the temperatureof incubation with the labelled substrate (12 or 23°C).Sensitivity of sucrose uptake to NEM only appeared with vesiclesthat had been stored in the presence of DTT, and if the pretreatmentwas run at 23°C. The temperature of incubation with labelledsucrose did not affect NEM sensitivity. The NEM sensitivityof valine uptake was not affected in the same way as sucroseuptake by the temperature of preincubation, showing that theeffects observed were specific for a given transporter. Underconditions which normally inhibit sucrose uptake, addition ofsucrose during NEM pretreatment protected the sucrose transporteragainst NEM inhibition. Key words: Sugar transport, plasma membrane, differential labelling, thiol reagents 相似文献
13.
Yan Liu Qing Wei Shu-Jun Wang Hong Liu Ning-Yi Zhou 《Applied microbiology and biotechnology》2010,87(1):281-287
Pseudomonas sp. strain NyZ402, a native soil organism that grows on para-nitrophenol (PNP), was genetically engineered for the simultaneous degradation of methyl parathion (MP) and ortho-nitrophenol (ONP) by integrating mph (methyl parathion hydrolase gene) from Pseudomonas sp. strain WBC-3 and onpAB (ONP 2-monooxygenase and ONP o-benzoquinone reductase genes) from Alcaligenes sp. strain NyZ215 into the genome of strain NyZ402. Methyl parathion hydrolase (MPH), ONP 2-monooxygenase (OnpA) and o-benzoquinone reductase (OnpB) were constitutively expressed in the engineered strain NyZ-MO. Strain NyZ-MO was free of exogenous
antibiotic resistance gene markers and the introduced genes were genetically stable. Degradation experiments showed that strain
NyZ-MO could utilize MP or ONP as the sole carbon and energy source, and mineralize 0.1 mM MP–0.1 mM ONP simultaneously. This
method may serve as a useful strategy for the construction of engineered strains in the degradation of multiple environmental
pollutants. 相似文献
14.
Mitochondrial NADH:ubiquinone-reductase (Complex I) catalyzes proton translocation into inside-out submitochondrial particles. Here we describe a method for determining the stoichiometric ratio
(n) for the coupled reaction of NADH oxidation by the quinone acceptors. Comparison of the initial rates of NADH oxidation and alkalinization of the surrounding medium after addition of small amounts of NADH to coupled particles in the presence of Q1 gives the value of n = 4. Thermally induced deactivation of Complex I [1,2] results in complete inhibition of the NADH oxidase reaction but only partial inhibition of the NADH:Q1-reductase reaction. N-Ethylmaleimide (NEM) prevents reactivation and thus completely blocks the thermally deactivated enzyme. The residual NADH:Q1-reductase activity of the deactivated, NEM-treated enzyme is shown to be coupled with the transmembraneous proton translocation (n = 4). Thus, thermally induced deactivation of Complex I as well as specific inhibitors of the endogenous ubiquinone reduction (rotenone, piericidin A) do not inhibit the proton translocating activity of the enzyme. 相似文献
15.
The initial rates of ATP synthesis catalyzed by tightly coupled Paracoccus denitrificans plasma membrane were measured. The reaction rate was hyperbolically dependent on the substrates, ADP and inorganic phosphate
(Pi). Apparent K
m values for ADP and Pi were 7–11 and 60–120 μM, respectively, at saturating concentration of the second substrate (pH 8.0, saturating Mg2+). These values were dependent on coupling efficiency. The substrate binding in the ATP synthesis reaction proceeds randomly:
K
m value for a given substrate was independent of the concentration of the other one. A decrease of electrochemical proton gradient
by the addition of malonate (when succinate served as the respiratory substrate) or by a decrease of steady-state level of
NADH (when NADH served as the respiratory substrate) resulted in a proportional decrease of the maximal rates and apparent
K
m values for ADP and Pi (double substitution, ping-pong mechanism). The kinetic scheme for ATP synthesis was compared with that described previously
for the proton-translocating ATP hydrolysis catalyzed by the same enzyme preparation (T. V. Zharova and A. D. Vinogradov (2006)
Biochemistry, 45, 14552–14558). 相似文献
16.
In a previous study, we characterized Cd–Hg interactions for uptake in human intestinal Caco-2 cells. We pursued our investigations
on metal uptake from metal mixtures, focusing on the effects of Hg on cellular homeostasis. A 4-fold higher equilibrium accumulation
value of 0.3 μmol/L 203Hg was measured in the presence of 100 μmol/L unlabeled Hg in the serum-free exposure medium without modification in the initial
uptake rate. This phenomenon was eliminated at 4∘C. Mercury induced an increase in tritiated water and [3H]mannitol uptakes for exposure times greater than 20 min. Incubations for 20 min and 30 min with 100 μmol/L Hg and 2 mmol/L
N-ethylmaleimide (NEM) resulted in a 34% and 50% reductions in cellular thiol staining, respectively, with additive effects.
Lactate dehydrogenase leakage and live/dead assays confirmed the maintenance of cell membrane integrity in Hg- or NEM-treated
cells. We conclude that Hg may alter membrane permeability and increase cell volume without any loss in cell viability. This
phenomenon is sensitive to temperature and could involve Hg interaction with membrane thiols, possibly related to solute transport.
During metal uptake from metal mixtures, Hg may thus promote the uptake of other toxic metals by increasing cell volume and
consequently cell capacity.
Deceased 25 March 2004 相似文献
17.
The regulation of the voltage-activated chloride current conductance (G
Cl
) in toad skin was investigated by the use of the SH reagents N-ethylmaleimide (NEM) and p-chloro-mercuricbenzenesulfonic
acid PCMBS. This anion pathway is controlled by a voltage-sensitive gating regulator. Mucosal application of NEM decreased
the voltage-activation in a time and concentration dependent manner, half-maximal inhibition being exerted at a concentration
of 30 μm within 20 min. At concentrations higher than 100 μm, the voltage-activated G
Cl
was near-completely and irreversibly inhibited in less than 10 min. Resting, deactivated conductance was essentially unaffected.
NEM had no effect on active sodium transport (measured as I
sc
) under conditions, which fully dissipated the voltage-activated G
Cl
. After complete inhibition of the voltage-activated G
Cl
with NEM, chloride conductance could still be stimulated by CPT-cAMP as in control tissues. Under these conditions, NEM at
concentrations above 1 mm decreased G
Cl
reversibly. Mucosal application of PCMBS at 500 μm inhibited the activated conductance by 35%, which was slightly reversible. Inhibition of voltage-activated G
Cl
, which was observed after mucosal addition of the membrane-impermeable NEM analogue, eosin-5-maleimide, was completely reversible
after washout. This suggests that the binding site for the maleimide is not accessible from the external face of the apical
membrane. Brief application of NEM at lower concentrations (1–3 min, ≤100 μm) led to partial inhibition of G
Cl
, followed by occasionally complete recovery upon washout of NEM. Recovery of voltage-activated G
Cl
was progressively attenuated and eventually disappeared after subsequent brief applications of NEM. This could reflect recruitment
of permeation/control sites from a finite pool. The data are discussed in the frame of a working model for the voltage-activated
Cl−-pathway, that contains two principle components, i.e., an anion-selective permeation path which is controlled by regulatory
protein(s).
Received: 18 December 1996/Revised: 28 April 1997 相似文献
18.
N E Lofrumento F Zanotti A Pavone 《Bollettino della Società italiana di biologia sperimentale》1979,55(8):715-721
As already reported, it has been found that the gradient of protons, set up across the inner membrane during the Ca2+ uptake by rat liver mitochondria, can be completely reversed by the addition of NEM. Identical results have been obtained by following the energy dependent K+ uptake. In these last conditions, the rate of H+ efflux supported by succinate oxidation is greatly enhanced only when NEM is added after rotenone. It is proposed that the increased rate other than to the inhibition of Pi uptake, as suggested by Reynafarje and Lehninger, could also be ascribed to a further decrease in the energetic level of the membrane as well as to an increased rate of succinate-Pi exchange diffusion reaction induced by NEM. A possible direct effect of NEM on succinate oxidation has been also considered to account for the inhibition observed when it is added before rotenone. 相似文献
19.
A. Mark Dobbing Joo-Yeon Han A. G. Sykes 《Journal of biological inorganic chemistry》1998,3(6):620-626
Dithionite has been found to reduce directly (without mediators) the Escherichia coli R2 subunit of ribonucleotide reductase. With dithionite (∼10 mM) in large excess, the reaction at 25 °C is complete in ∼10 h.
Preparations of E. coli R2 have an FeIII
2 (met-R2) component in this work at ∼40% levels, alongside the fully active enzyme FeIII
2 . . . Tyr*, which has a tyrosyl radical at Tyr-122. In the pH range studied (7–8) the kinetics are biphasic. Rate laws for
both phases give [S2O4
2–] and not [S2O4
2–]1/2 dependencies, and saturation kinetics are observed for the first time in R2 studies. No dependence on pH was detected. The
kinetics (25 °C) of the first phase are reproduced in separate experiments using only met-R2, with association of S2O4
2– to met-R2, K=330 M–1, occurring prior to electron transfer, k
et=4.8×10–4 s–1, I=0.100 M (NaCl). The second phase assigned to the reaction of FeIII
2 . . . Tyr* with S2O4
2– gives K=800 M–1 and k
et=5.6×10–5 s–1. Bearing in mind the substantially smaller reduction potential for FeIII
2 compared to Tyr*, this is a quite remarkable finding, with implications similar to those already reported for the reaction
of R2 with hydrazine, but with additional information provided by the saturation kinetics. The similarity in rates for the
two phases (∼fourfold difference) suggests that reduction of FeIII
2 is occurring in both cases, and since S2O4
2– is involved a two-equivalent change is proposed with the formation of FeII
2 . . . Tyr* in the case of active R2. As a sequel to the second phase, intramolecular reduction of the strongly oxidising
Tyr* by the FeII
2 is rapid, and further decay of FeIIFeIII is also fast. There is no stable mouse met-R2 form, and the single-phase reaction with dithionite gives saturation kinetics
with K=208 M–1 and k
et=1.7±10–3 s–1. Mechanistic implications, including the applicability of a pathway for electron transfer via FeA, are considered.
Received: 25 February 1998 / Received: 20 August 1998 相似文献
20.
C. F. Shaw III. Libin He Amalia Muñoz M. Meral Savas Susan Chi Cynthia L. Fink Tong Gan David H. Petering 《Journal of biological inorganic chemistry》1997,2(1):65-73
The model alkylating agent N-ethylmaleimide (NEM) reacts reversibly at the metal-bound thiolates of Zn7MT and Cd7MT. An unprecedented feature of this reaction is that it approaches equilibrium and requires a large excess of NEM (>1 mM
for 3 μM protein) to drive it to completion. The complex kinetics of the reaction can be followed by monitoring the release
of bound metal ions using the metallochromic dyes Zincon (ZI) for Zn7MT and pyridylazoresorcinol for Cd7MT. An initial lag phase is followed by more rapid release of zinc ions. The observed pseudo-first-order rate constants for
the two phases are independent of the ZI and Zn7MT concentrations. The complex NEM concentration dependence of each phase,
k
f, obs=k
f
1+k
f
2 [NEM] and k
s, obs=k
s
1+k
s
2 [NEM],
demonstrates that the forward reactions are second order and the reverse reactions are first order. The alkylation can be
reversed using 2-mercaptoethanol to compete for the protein-bound NEM and regenerate the Zn-binding capability of alkylated
MT. An explanation of these observations, based on the reversibility of cysteine alkylation by NEM, was developed and tested.
The reactions of Cd7MT are less complete than those of Zn7MT and occur more slowly. 111Cd-NMR studies of the partially alkylated 111Cd7MT reveal that reaction with only four equivalents of NEM completely alters the cluster structure and eliminates the spectral
signatures of the α and β clusters, although very little cadmium has been removed from the protein. This finding substantiates
the proposed kinetic intermediate, a partially alkylated MT with complete or nearly complete retention of the metal ions,
and rules out the possibility of cooperative reactions at either cluster.
Received: 5 August 1996 / Accepted: 24 October 1996 相似文献