• 미생물학회지
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• 제29권5호
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• pp.278-283
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• 1991

Two amino acid residues ($Ala^{494}$ and $Ser^{495}$ of E. coli .gamma.-glutamylcysteine synthetase have been investigated whether they are the site of feedback inhibition by site specific mutagenesis. Single substitution of $serine^{495}$ (S495F), and double substitutions of alanine$^{494}$ and $serine^{495}$ (A494G-S495F) resulted in the inactivation of the .gamma.-glutamylcysteine synthetase activity. Substitution of $alanine^{494}$ with $glycine^{494}$ resulted in a higher level of feedback inhibition. These results suggest that $serine^{495}$ in .gamma.-glutamylcysteine synthetase is required for its catalytic acitvity and $alanine^{494}$ is presumably related to the feeback inhibition site.

• Journal of Microbiology and Biotechnology
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• 제11권5호
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• pp.838-844
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• 2001

The enzyme Fuc aldolase from Methanococcus jannaschii that catalyzes the aldol condensation of DHAP and L-lactaldehyde to give fuculose-1-phosphate was inactivated by DEP. The inactivation was pseudo first-order in the enzyme and DEP, which was biphasic. A pseudo second-order rate constant of 120$M^{-1}min^{-1}$ was obtained at pH 6.0 and $25{\circ}C$. Quantifying the increase in absorbance at 240nm showed that four histidine residues per subunit were modified during the nearly complete inactivation. The statistical analysis and the time course of the modification suggested that two or three histidine residues were essential for activity. The rate of inactivation was dependent on the pH, and the pH inactivation data implied the involvement of the amino acid residue with a $pK_a$ value of 5.7. Fuc aldolase was protected against DEP inactivation by DHAP, indicating that the histidine residues were located at the active site of Fuc aldolase. DL-Glyceraldehyde, as an alternative substrate to L-lactaldehyde, showed no specific protection for the Fuc aldolase.

• 한국미생물·생명공학회지
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• 제34권2호
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• pp.121-128
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• 2006

Bacillus alcalophilus AX2000으로부터 xylanase를 정제한 후 효소의 활성부위를 조사하기 위하여 여러 가지 화학수식제를 사용하여 효소활성의 저해도를 측정하였다. 여러 가지 화학 수식제 중에서 carbodiimide와 N-bromosuccinimide가 효소 활성을 완전히 저해시켜 glutamic acid또는 aspartic acid 잔기와 tryptophan 잔기가 효소의 활성부위에 관여하리라 추측되었다. 각각의 경우에 효소 실활은 수식제의 첨가농도에 따라 pseudo first-order kinetics 양식을 보여주었으며, carbodiimide와 N-bromosuccinimide는 각각 비경쟁적 저해와 경쟁적 저해방식을 나타내었다. 기질첨가에 의한 효소활성 보호실험을 통하여 tryptophan 잔기가 기질결합부위라 판단 되었다. 효소 실활속도의 분석에 의해 효소활성에는 2개의 glutamic acid 또는 aspartic acid 잔기와 1개의 tryptophan 잔기가 관여하는 것으로 나타났다.

• BMB Reports
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• 제32권5호
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• pp.515-520
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• 1999

Incubation of two types of glutamate dehydrogenase isoproteins from bovine brain with the arginine-specific dicarbonyl reagent phenylglyoxal resulted in a biphasic loss of enzyme activity. Reaction of the glutamate dehydrogenase isoproteins with phenylglyoxal caused a rapid loss of 53~62% of the enzyme activities and modification of two residues of arginine per enzyme subunit. Prolonged incubation of the glutamate dehydrogenase isoproteins with phenylglyoxal resulted in the modification of an additional four residues of arginine per enzyme subunit without further loss of the residual activities. Partial protection against inactivation was provided by the coenzyme NADH or substrate 2-oxoglutarate. The most marked decrease in the rate of inactivation was observed by the combined addition of NADH and 2-oxoglutarate, suggesting that the first two modified arginine residues are in the vicinity of the catalytic site. However, inactivation of the glutamate dehydrogenase isoproteins by phenylglyoxal appears to be partial with approximately 40% activity remained after an extended reaction time with excess reagent, suggesting that the modified arginine residues may not be directly involved in catalysis. The lack of complete protection by substrates also suggest the possibility that the modified arginine residues are not directly involved at the active site, and the partial loss of activity by the modification of arginine residues may be due to a conformational change. There were no significant differences between the two glutamate dehydrogenase isoproteins in sensitivities to inactivation by phenylglyoxal, indicating that the microenvironmental structures of the glutamate dehydrogenase isoproteins are very similar to each other.

• 생명과학회지
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• 제15권4호
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• pp.633-640
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• 2005

Bacillus pumilus TX703으로부터 xylanase를 정제한 후 효소의 활성부위를 조사하기 위하여 여러 가지 화학수식제를 사용하여 효소활성의 저해도를 측정하였다. 여러 가지 화학수식제 중에서 carbodiimide와 N-bromosuccinimide가 효소활성을 완전히 저 해시 켜 glutamic acid 또는 aspartic acid 잔기와 tryptophan 잔기가 효소의 활성부위에 관여하리라 추측되었다. 각각의 경우에 효소 실활은 수식제의 첨가농도에 따라 pseudo first-order kinetics 양식을 보여주었으며, car-bodiimide와 N-bromosuccinimide는 각각 비경쟁적 저해와 경쟁적 저해방식을 나타내었다. 기질첨가에 의한 효소활성 보호실험을 통하여 tryptophan 잔기가 기질결합부위라 판단되었다. 효소실활속도의 분석에 의해 효소활성에는 2개의 glutamic acid 또는 aspartic acid 잔기와 1개의 tryptophan 잔기가 관여하는 것으로 나타났다.

• BMB Reports
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• 제29권5호
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• pp.436-441
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• 1996

The palmitoyl acyl carrier protein (ACP) specific thioesterase (EC 3.1.2.14) from Iris pseudoacorus was purified and characterized. The thioesterase which was very unstable in relatively high salt concentrations was eluted using a co-gradient of Triton X-100 and low concentration of KCl or Na-phosphate from Q-Sepharose, DEAE-Sepharose, and hydroxyapatite chromatography. SDS-PAGE analysis showed a single band with a molecular weight of 35,000. The native molecular weight of approximately 37,000 was estimated by Sephacryl S-200 chromatography, indicating that the enzyme is a monomer. The thioesterase activity was inhibited about 75% and 50% by N-ethylmaleimide (2 mM) and phenylmethylsulfonyl fluoride (2 mM). respectively. The N-ethylmaleimide-inactivation was protected by sodium palmitate but the inactivation with phenylmethylsulfonyl fluoride was not protected. Oxidation of thiols by 2 mM 5.5'-dithio-bis-(2-nitrobenzoic acid) resulted in 65% inactivation of the enzyme. These results suggest that a cysteinyl residue is essential to the catalytic reaction of the enzyme. The enzyme activity was increased by sodium citrate and also by $Cu^{2+}$

• 미생물학회지
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• 제29권5호
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• pp.301-306
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• 1991

Extracellular fructosyl transferase from Aureobasidium pullulans C-23 was characterized. The molecular weight of the isolated enzyme was determined to be approximately 170,000 by SDS polyacrylamide gel electrophoresis. The enzyme has the pI value of about 3.7. The enzyme was almost completely inhibited by 5mM $Hg^{2+}$ , but was not significantly affected by other cations tested. The enzyme was inactivated by treatment of tryptophan-specific reagent N-bromo- succinimide and tyrosine-specific reagent iodine. The substrate sucrose showed protective effect on the inactivation of the enzyme by the both reagents. These results suggest that tryptophan and tyrosine residues are probably located at or near active site of the enzyme.

• BMB Reports
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• 제37권2호
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• pp.260-267
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• 2004

Transducin (T), the heterotrimeric guanine nucleotide binding protein in rod outer segments, serves as an intermediary between the receptor protein, rhodopsin, and the effector protein, cGMP phosphodiesterase. Labeling of T with dansyl chloride (DnsCl) inhibited its light-dependent guanine nucleotide binding activity. Conversely, DnsCl had no effect on the functionality of rhodopsin. Approximately 2-3 mol of DnsCl were incorporated per mole of T. Since fluoroaluminate was capable of activating DnsCl-modified T, this lysine-specific labeling compound did not affect the guanine nucleotide-binding pocket of T. However, the labeling of T with DnsCl hindered its binding to photoexcited rhodopsin, as shown by sedimentation experiments. Additionally, rhodopsin completely protected against the DnsCl inactivation of T. These results demonstrated the existence of functional lysines on T that are located in the proximity of the interaction site with the photoreceptor protein.

• BMB Reports
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• 제35권2호
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• pp.178-185
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• 2002

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA ($GenBank^{TM}$ U63633) was cloned. Site-specific mutagenesis was performed to introduce mutations at the conserved cysteine $Cys^{50}$, $Cys^{83}$, and $Cys^{230}$, and $lys^{81}$ residues. In accordance with the human AdoMetDC, the C50A and C230A mutagenesis had minimal effect on catalytic activity, which was further supported by DTNB-mediated inactivation and reactivation. However, unlike the human AdoMetDC, the $Cys^{50}$ and $Cys^{230}$ mutants were much more thermally unstable than the wild type and other mutant AdoMetDC, suggesting the structural significance of cysteines. Furthermore, according to a circular dichroism spectrum analysis, the $Cys^{50}$ and $Cys^{230}$ mutants show a higher a-helix content and lower coiled-coil content when compared to that of wild type and the other mutant AdoMetDC. Also, the three-dimensional structure of Arabidopsis thaliana AdoMetDC could further support all of the data presented here. Summarily, we suggest that the $Cys^{50}$ and $Cys^{230}$ residues are structurally important.

• Animal cells and systems
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• 제1권4호
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• pp.583-587
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• 1997

Incubation of an NADPH-dependent succinic semialdehyde reductase from bovine brain with N-bromosuccinimide (NBS) resulted in a time-dependent loss of enzyme activity. The inactivation followed pseudo-first-order kinetics with the second-order rate constant of $6.8\times{10}^3$ $M^-1$ $min^{-1}$. The inactivation was prevented by preincubation of the enzyme with substrate succinic semialdehyde, but not with coenzyme NADPH. There was a linear relation-ship between oxindole formation and the loss of enzyme activity. Spectro-photometric studies indicated that about one oxindole group per molecule of the enzyme was formed following complete loss of enzymatic activity. It is suggested that the catalytic function of succinic semialdehyde reductase is modulated by binding of NBS to a specific tryptophan residue at or near the substrate binding site of the enzyme.