• 생명과학회지
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• 제9권2호
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• pp.146-152
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• 1999

본 연구에서는 대장균 트립토판 중합효소(tryptophan synthase) $\alpha$ 소단위체의 폴딩에 있어 24번 잔기의 역할을 보고자 하였다. 24번 잔기인 트레오닌이 메티오닌, 알라닌, 세린, 류신 또는 리신으로 치환된 단백질를 대장균내에서 과량 발현시켜 수용성 구조의 양과 비수용성인 inclusion body양을 조사하였다. 그 결과 메티오닌이나 류신으로 치환된 $\alpha$ 소단위체는 트레오닌이 있는 소단위체와 마찬가지로 수용성 구조가 대부분을 차지하였다. 반면, 세린이나 알라닌 또는 리신으로 치환된 단백질들은 많은 양이 inclusion body로 발현되었다. 이 결과는 트립토판 중합효소 $\alpha$ 소단위체의 폴딩에 있어서 24번 잔기가 관여하고 있음을 제시하고 있다.

• 생명과학회지
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• 제9권6호
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• pp.733-736
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• 1999

대장균 트립토판 중합요소 (tryptophan synthase) $\alpha$ 소단위체의 24번 잔기인 트레오닌이 메티오닌, 알라닌 또는 세린으로 치환되고 잔기 139에 트립토판이 있는 단백질를 정제하여, 여러가지 요소 농도에 대한 트립토판 형광값의 변화를 측정하여 변성곡선을 얻었다. 이들 단백질들은 모두 F139W 단백질에 비해 구조형성 성질에 큰 변화를 일으켰으며, 잔기 24번은 구조형성에 중요한 것으로 추정된다.

• Bulletin of the Korean Chemical Society
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• 제22권1호
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• pp.68-76
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• 2001

We have employed chemical modification to identify amino acids essential for the catalytic activity of alliinase (EC 4.4.1.4) from garlic (Allium sativum). Alliinase degrades S-alkyl-L cysteine sulfoxides, causing the characteristic odor of garlic. The activity of alliinase was rapidly and completely inactivated by N-bromosuccinimide(NBS) and slightly decreased by succinic anhydride and N-acetylimidazole. These results indicate that tryptophanyl, lysyl, and tyrosyl residues play an important role in enzyme catalysis. The reaction of alliinase with NBA yielded a characteristic decrease in both the absorbance at 280 nm and the intrinsic fluorescence at 332 nm with increasing reagent concentration of NBS, consistent with the oxidation of tryptophan residues. Kinetic analysis, fluorometric titration of tryptophans and correlation to residual alliinase activity showed that modification of only one residue present on alliinase led to complete inhibition of alliinase activity. To identify this essential tryptophan residue, we employed chemical modification by NBS in the presence and absence of the protecting substrate analogue, S-ethyl-L-cysteine (SEC) and N-terminal sequence analysis of peptide fragment isolated by reverse phase-HPLC. A fragment containing residues 179-188 was isolated. We conclude that Trp182 is essential for alliinase activity.

• 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.

• Bulletin of the Korean Chemical Society
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• 제34권3호
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• pp.963-966
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• 2013

• Bulletin of the Korean Chemical Society
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• 제9권4호
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• pp.202-206
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• 1988

In order to investigate the oligomerization state of the plasma membrane proteolipid apoprotein purified from the bovine kidney, fluorescence polarization experiment was carried out in the two different solvent systems, i.e., water and organic solvent(chloroform-methanol). The molecular volumes of the proteins estimated from the Perrin equation, were to be 45,258$A^3$ and 17,608$A^3$ in water and organic solvent, respectively. These values indicate that a trimerization is possibly occurring in the aqueous environment. As an auxiliary experiment for the calculation of the molecular volume using Perrin equation, fluorescence quenching constants ($K_q$) with the quencher acrylamide and fluorescence lifetimes (${\tau}_F$) of the intrinsic fluorophore tryptophan residue were estimated in the two different solvent systems. $K_q$ in water was 18.21$M^{-1}$ and it was 46.24$M^{-1}$ in organic solvent. Fluorescence lifetimes of tryptophan residue were calculated to be 2.80 nsec. in water and 3.81 nsec. in organic solvent, respectively.

• BMB Reports
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• 제29권4호
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• pp.321-326
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• 1996

Spinach glycolate oxidase was subjected to a series of chemical modifications aimed at identifying amino acid residues essential for catalytic activity. The oxidase was reversibly inactivated by treatment with pyridoxal 5'-phosphate (PLP). The inactivation by PLP was accompanied by the appearance of an absorption peak of around 430 nm, which was shifted to 325 nm upon reduction with $NaBH_4$. After reduction, the PLP-treated oxidase showed a fluorescence spectrum with a maximum of around 395 nm by exciting at 325 nm. The substrate-competitive inhibitors oxalate and oxaloacetate provided protection against inactivation of the oxidase by PLP. These results suggest that PLP inactivates the enzyme by fonning a Schiff base with lysyl residue(s) at an active site of the oxidase. The enzyme was also inactivated by tryptophan-specific reagent N-bromosuccinimide (NBS). However, competitive inhibitors oxalate and oxaloacetate could not protect the oxidase significantly against inactivation of the enzyme by NBS. The results implicate that the inactivation of the oxidase by NBS is not directly related to modification of the tryptophanyl residue at an active site of the enzyme. Treatments of the oxidase with cysteine-specific reagents iodoacetate, silver nitrate, and 5,5'-dithiobis-2-nitrobenzoic acid did not affect significantly the activity of the enzyme.

• 한국미생물·생명공학회지
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• 제25권2호
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• pp.173-177
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• 1997

Glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis was modified with various chemical modifiers to determine the active sites of the enzyme. Treatment of the enzyme with group-specific reagents diethylpyrocarbonate, N-bromosuccinimide, or carbodiimide resulted in complete loss of enzyme activity, which shows histidine, tryptophan, and glutamic acid or aspartic acid residues are at or near the active site. In each case, inactivation followed pseudo first-order kinetics. Inclusion of glycerol-3-phosphate and/or CTP prevented the inactivation, indicating the presence of tryptophan and glutamic acid or aspartic acid residues at the substrate binding site. Analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a two histidine residues, single tryptophan residue, and two glutamic acid or aspartic acid residues.

• BMB Reports
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• 제32권3호
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• pp.294-298
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• 1999

Myo-inositol monophosphate phosphatase is a key enzyme in the phosphoinositide cell-signaling system. Incubation of myo-inositol monophosphate phosphatase from porcine 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 $3.8{\times}10^3\;M^{-1}min^{-1}$. The time course of the reaction was significantly affected by the substrate myo-inositol-1-phosphate, which afforded complete protection against the loss of catalytic activity. Spectrophotometric studies indicated that about one oxindole group per molecule of enzyme was formed following complete loss of enzymatic activity. It is suggested that the catalytic function of myo-inositol monophosphate phosphatase is modulated by the binding of NBS to a specific tryptophan residue at or near the substrate binding site of the enzyme.

• 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.