Dihydroflavonol 4-reductase (DFR), a member of the short-chain dehydrogenase family, catalyzes the last common step in the biosynthesis of flavan-3-ols and condensed tannins. Initial rates of DFR were measured by moni...Dihydroflavonol 4-reductase (DFR), a member of the short-chain dehydrogenase family, catalyzes the last common step in the biosynthesis of flavan-3-ols and condensed tannins. Initial rates of DFR were measured by monitoring the 340-nm absorbance decrease resulting from the joint consumption of dihydroquercetin (DHQ) and NADPH, as a function of pH, temperature and ionic strength. At pH 6.5 and 30o C, substrate inhibition was observed above 30 μM DHQ. At lower/non-inhibitory DHQ concentrations, NADP+ behaves as a competitive inhibitor with respect to NADPH and as a mixed inhibitor with respect to DHQ, which supports a sequential ordered mechanism, with NADPH binding first and NADP+ released last. Binding-equilib-rium data obtained by means of the chromatographic method of Hummel and Dreyer at pH 7.5 and by isothermal calorimetric titration at pH 6.5 led to the conclusion that ligands of the apoenzyme included NADPH, NADP+ and DHQ. The mechanism which best accounts for substrate inhibition at pH 6.5 in the absence of product involves the formation of a binary non-productive E.DHQ complex. Thus, a productive ternary complex cannot be formed when DHQ binds first. This mechanism of inhibition may prevent the accumulation of unstable leucoanthocyanidins within cells.展开更多
Glutathione peroxidase (GPx, EC1.11.1.9), an important anti-oxidative selenoenzyme, can catalyze the reduction of harmful hydroperoxides with concomitant glutathione, thereby protecting cells and other biological issu...Glutathione peroxidase (GPx, EC1.11.1.9), an important anti-oxidative selenoenzyme, can catalyze the reduction of harmful hydroperoxides with concomitant glutathione, thereby protecting cells and other biological issues against oxidative damage. It captures considerable interest in redesign of its function for either the mechanism study or the pharmacological development as an antioxidant. In order to de- velop a general strategy for specifically targeting and operating selenium in active sites of enzymes, the catalytically essential residue selenocysteine (Sec) was first successfully bioincorporated into the catalytic center of subtilisin by using an auxotrophic expression system. The studies of the catalytic activity and the steady-state kinetics demonstrated that selenosubtilisin is an excellent GPx-like bio- catalyst. In comparison with the chemically modified method, biosynthesis exhibits obvious advan- tages: Sec could be site-directly incorporated into active sites of enzymes to overcome the non-speci- ficity generated by chemical modification. This study provides an important strategy for specifically targeting and operating selenium in the active site of an enzyme.展开更多
Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH4)SO4 precipitation, extraction of palm leaf colo...Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH4)SO4 precipitation, extraction of palm leaf colored compounds and consecutive chromatography on Phenyl-Sepharose, TSK-Gel DEAE-5PW and Superdex-200. The novel peroxidase was characterized as having a molecular weight of 48.2 ± 3.0 kDa and an isoelectric point pI 5.4 ± 0.1. The enzyme forms dimers in solution with approximate molecular weight of 92 ± 2 kDa. Here we investigated the steady-state kinetic mechanism of the H2O2-supported oxidation of different organic substrates by RPTP. The results of the analysis of the initial rates vs. H2O2 and reducing substrate concentrations were seen to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach used expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters KmH2O2, KmAH2, kcat, KSIH2O2, KSIAH2 and of the microscopic rate constants k1 and k3 of the shared three-step peroxidase catalytic cycle. Furthermore, the concentration and time-dependences and the mechanism of the suicide inactivation of RPTP by hydrogen peroxide were studied kinetically with guaiacol as co-substrate. The turnover number (r) of H2O2 required to complete the inactivation of the enzyme was 2154 ± 100 and the apparent rate constants of catalysis 185 s–1 and 18 s–1.展开更多
The steady-state kinetics for complete oxidation of benzene over has been investigated by the external recycling reactor. The kinetics equation was described by the L-H model of adsorption of benzene and oxygen with t...The steady-state kinetics for complete oxidation of benzene over has been investigated by the external recycling reactor. The kinetics equation was described by the L-H model of adsorption of benzene and oxygen with the inhibition of carbon dioxide. The parameters of the kinetics model were estimated by the method of orthogonal design. The heats of adsorption of benzene, oxygen and carbon dioxide were determined by the method of gas-adsorption chromatography. The details of oscillations of complete oxidation of benzene were investigated.展开更多
Many enzymes which catalyze the conversion of large substrates are made of several structural domains belonging to the same polypeptide chain. Transfer RNA (tRNA), one of the substrates of the multidomain aminoacyl-tR...Many enzymes which catalyze the conversion of large substrates are made of several structural domains belonging to the same polypeptide chain. Transfer RNA (tRNA), one of the substrates of the multidomain aminoacyl-tRNA synthetases (aaRS), is an L-shaped molecule whose size in one dimension is similar to that of its cognate aaRS. Crystallographic structures of aaRS/tRNA complexes show that these enzymes use several of their structural domains to interact with their cognate tRNA. This mini review discusses first some aspects of the evolution and of the flexibility of the pentadomain bacterial glutamyl-tRNA synthetase (GluRS) revealed by kinetic and interaction studies of complementary truncated forms, and then illustrates how stable analogues of aminoacyl-AMP intermediates have been used to probe conformational changes in the active sites of Escherichia coli GluRS and of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) of Pseudomonas aeruginosa.展开更多
We report a comparative study on photo-crystallization in a-Se95 Te5 and a-Se95In5 alloys. The photo-crystallization is achieved by a shining white line on the thin films of these alloys in vacuum for different exposu...We report a comparative study on photo-crystallization in a-Se95 Te5 and a-Se95In5 alloys. The photo-crystallization is achieved by a shining white line on the thin films of these alloys in vacuum for different exposure times. The results indicate that photo-crystallization is fast in a-Se95In5 alloy as compared to a-Se95 Te5 alloy. This is explained in terms of lower thermal stability of a-Se95In5 alloy as compared to a-Se95 Te5 alloy.展开更多
文摘Dihydroflavonol 4-reductase (DFR), a member of the short-chain dehydrogenase family, catalyzes the last common step in the biosynthesis of flavan-3-ols and condensed tannins. Initial rates of DFR were measured by monitoring the 340-nm absorbance decrease resulting from the joint consumption of dihydroquercetin (DHQ) and NADPH, as a function of pH, temperature and ionic strength. At pH 6.5 and 30o C, substrate inhibition was observed above 30 μM DHQ. At lower/non-inhibitory DHQ concentrations, NADP+ behaves as a competitive inhibitor with respect to NADPH and as a mixed inhibitor with respect to DHQ, which supports a sequential ordered mechanism, with NADPH binding first and NADP+ released last. Binding-equilib-rium data obtained by means of the chromatographic method of Hummel and Dreyer at pH 7.5 and by isothermal calorimetric titration at pH 6.5 led to the conclusion that ligands of the apoenzyme included NADPH, NADP+ and DHQ. The mechanism which best accounts for substrate inhibition at pH 6.5 in the absence of product involves the formation of a binary non-productive E.DHQ complex. Thus, a productive ternary complex cannot be formed when DHQ binds first. This mechanism of inhibition may prevent the accumulation of unstable leucoanthocyanidins within cells.
基金the National Natural Science Foundation of China (Grant Nos 20534030 and 20725415)National Basic Research Program of China (Grant No. 2007CB808006) Innovative Research Team in University of China (Grant No. IRT0422)
文摘Glutathione peroxidase (GPx, EC1.11.1.9), an important anti-oxidative selenoenzyme, can catalyze the reduction of harmful hydroperoxides with concomitant glutathione, thereby protecting cells and other biological issues against oxidative damage. It captures considerable interest in redesign of its function for either the mechanism study or the pharmacological development as an antioxidant. In order to de- velop a general strategy for specifically targeting and operating selenium in active sites of enzymes, the catalytically essential residue selenocysteine (Sec) was first successfully bioincorporated into the catalytic center of subtilisin by using an auxotrophic expression system. The studies of the catalytic activity and the steady-state kinetics demonstrated that selenosubtilisin is an excellent GPx-like bio- catalyst. In comparison with the chemically modified method, biosynthesis exhibits obvious advan- tages: Sec could be site-directly incorporated into active sites of enzymes to overcome the non-speci- ficity generated by chemical modification. This study provides an important strategy for specifically targeting and operating selenium in the active site of an enzyme.
文摘Royal palm tree peroxidase (RPTP) has been isolated to homogeneity from leaves of Roystonea regia palm trees. The enzyme purification steps included homogenization, (NH4)SO4 precipitation, extraction of palm leaf colored compounds and consecutive chromatography on Phenyl-Sepharose, TSK-Gel DEAE-5PW and Superdex-200. The novel peroxidase was characterized as having a molecular weight of 48.2 ± 3.0 kDa and an isoelectric point pI 5.4 ± 0.1. The enzyme forms dimers in solution with approximate molecular weight of 92 ± 2 kDa. Here we investigated the steady-state kinetic mechanism of the H2O2-supported oxidation of different organic substrates by RPTP. The results of the analysis of the initial rates vs. H2O2 and reducing substrate concentrations were seen to be consistent with a substrate-inhibited Ping-Pong Bi-Bi reaction mechanism. The phenomenological approach used expresses the peroxidase Ping-Pong mechanism in the form of the Michaelis-Menten equation and affords an interpretation of the effects in terms of the kinetic parameters KmH2O2, KmAH2, kcat, KSIH2O2, KSIAH2 and of the microscopic rate constants k1 and k3 of the shared three-step peroxidase catalytic cycle. Furthermore, the concentration and time-dependences and the mechanism of the suicide inactivation of RPTP by hydrogen peroxide were studied kinetically with guaiacol as co-substrate. The turnover number (r) of H2O2 required to complete the inactivation of the enzyme was 2154 ± 100 and the apparent rate constants of catalysis 185 s–1 and 18 s–1.
基金Supported by the National Sciences Fundation of China.
文摘The steady-state kinetics for complete oxidation of benzene over has been investigated by the external recycling reactor. The kinetics equation was described by the L-H model of adsorption of benzene and oxygen with the inhibition of carbon dioxide. The parameters of the kinetics model were estimated by the method of orthogonal design. The heats of adsorption of benzene, oxygen and carbon dioxide were determined by the method of gas-adsorption chromatography. The details of oscillations of complete oxidation of benzene were investigated.
文摘Many enzymes which catalyze the conversion of large substrates are made of several structural domains belonging to the same polypeptide chain. Transfer RNA (tRNA), one of the substrates of the multidomain aminoacyl-tRNA synthetases (aaRS), is an L-shaped molecule whose size in one dimension is similar to that of its cognate aaRS. Crystallographic structures of aaRS/tRNA complexes show that these enzymes use several of their structural domains to interact with their cognate tRNA. This mini review discusses first some aspects of the evolution and of the flexibility of the pentadomain bacterial glutamyl-tRNA synthetase (GluRS) revealed by kinetic and interaction studies of complementary truncated forms, and then illustrates how stable analogues of aminoacyl-AMP intermediates have been used to probe conformational changes in the active sites of Escherichia coli GluRS and of the nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) of Pseudomonas aeruginosa.
文摘We report a comparative study on photo-crystallization in a-Se95 Te5 and a-Se95In5 alloys. The photo-crystallization is achieved by a shining white line on the thin films of these alloys in vacuum for different exposure times. The results indicate that photo-crystallization is fast in a-Se95In5 alloy as compared to a-Se95 Te5 alloy. This is explained in terms of lower thermal stability of a-Se95In5 alloy as compared to a-Se95 Te5 alloy.
