• 생명과학회지
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• 제10권2호
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• pp.210-217
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• 2000

A kinetic profile of the catalytic domain of stromelysin-1 (SCD) using the fluorescent peptide substrate has been determined by the stopped-flow technique. The pH profile has a pH optimum of about 5.5 with an extended shoulder above pH 7. Three pKa values, 5.0, 5.7, and 9.8 are found for the free enzyme state and two pH independent Kcat/Km values of 4.1$\times$104 M-1 s-1 and 1.4$\times$104 M-1 s-1 at low and high pH, respectively. The profile is quite different in shape with other MMP family which has been reported, having broad pH optimum with two pKa values. The substrate specificity of SCD towards fluorescent heptapeptide substrates has been also examined by thin layer chromatography. The cleavage sites of the substrates have been identified using reverse-phase HPLC method.SCD cleaves Dns-PLA↓L↓WAR and Dns-PLA↓L↓FAR at two positions. However, the Dns-PLA↓LRAR, Dns-PLE↓LFAR, adn Dns-PLSar↓LFAR are cleaved exclusively at one bond. The double cleavages of Dns-PLALWAR and Dns-PLALFAR by SCD are in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis agrees with the structural data that the S1' pocket of SCD is deeper than that of matriysin. The differences observed between SCD and matrilysin may form the basis of understanding the structural relationships and substrate specificities of the MMP family in vivo.

• BMB Reports
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• 제29권3호
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• pp.210-214
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• 1996

The pH dependence of the kinetic parameters of mutant O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium LT-2 has been determined in order to obtain information on the chemical mechanism. The initial velocity pattern obtained by varying the concentrations of OAS at several fixed concentrations of TNB, shows an intersection on the left of the ordinate at pH 7.0, indicating that the kinetic mechanism is a sequential mechanism in which substrate inhibition by OAS is observed while the wild type enzyme showed a ping pong mechanism. The values of $V/E_t$, $V/K_{OAS}E_{t}$ and $V/K_{TNB}E_{t}$ decreased by about 68%, 14% and 16% as compared with the wild type enzyme. The $V/K_{OAS}E_{t}$ is a pK of 6.5 on the acid side of the pH profile, and the $V/K_{TNB}$ is pH independent. As compared with the wild type enzyme, the pKs in the V/K profiles are shifted, reflecting that binding of the cofactor in free E:OAS is less asymmetric.

• 생명과학회지
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• 제12권1호
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• pp.32-42
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• 2002

합성된 hexanoylthiocholine을 기질로 하여 butyrylcholinesterase와의 반응을 연구하였다. 기질의 농도 변화에 따른 초기반응속도 관찰을 통해 아실기의 탄소수가 증가함에 따라 반응성은 감소하나 $K_{m}$ 값은 0.140(mM)으로 더 강한 ES 복합체를 형성함을 알 수 있었다. Hexanoylthio-choline의 촉매화된 acetylcholinesterase 가수분해에 대한 pH-V/K profile에서 p $K_{a}$ 값 4.974$\pm$0.02을 얻었다. 이는 최근 문헌의 보고와 상통하는 것으로 p $K_{a}$ =6.2~6.4를 갖는 잔류물의 기본형에 활성이 의존하는 것으로부터 하나의 잔류물 또는 p $K_{a}$ =4.7~5.0을 갖는 잔류물들의 촉매작용으로 계통적인 자리밀림을 보여준다. 이는 촉매화된 BChE의 활성영역 esteratic site 주변에 긴 사슬 아실기의 가수분해에 관여하는 새로운 활성영역이 존재함을 밝히는 증거이다. 분자 조형은 기질의 아실기의 탄소수에 따라 acetylcholinesterase에 의해 표현되어지는 반응과정의 변화의 합리성을 제공한다. 본 연구에서는 한국과학기술원 도핑컨트롤센터와 연계하여 acetylcholinesterase와 기질인 acylthiocholine과의 입체적으로 둘러싸인 acyl-binding site를 분자 조형하고자 노력 중에 있다.

• Bulletin of the Korean Chemical Society
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• 제30권8호
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• pp.1724-1728
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• 2009

Severe acute respiratory syndrome coronavirus (SARS-CoV) helicase separates the double-stranded nucleic acids using the energy from ATP hydrolysis. We have measured ATPase activity of SARS-CoV helicase in the presence of various types of nucleic acids. Steady state ATPase analysis showed that poly(U) has two-times higher turnover number than poly(C) with lower Michaelis constant. When M13 single-stranded DNA is used as substrate, the Michaelis constant was about twenty-times lower than poly(U), whereas turnover numbers were similar. However, stimulation of ATPase activity was not observed in the presence of double-stranded DNA. pH dependent profiles of ATP hydrolysis with the helicase showed that the optimal ATPase activities were in a range of pH 6.2 ~ 6.6. In addition, ATP hydrolysis activity assays performed in the presence of various divalent cations exhibited that $Mg^{2+}$ stimulated the ATPase activity with the highest rate and $Mn^{2+}$ with about 40% rate as compared to the $Mg^{2+}$.

• Bulletin of the Korean Chemical Society
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• 제34권6호
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• pp.1800-1808
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• 2013

Dual-responsive amphiphilic block copolymers were synthesized by combining enzymatic ring-opening polymerization (eROP) of ${\varepsilon}$-caprolactone (CL) and ATRP of N,N-dimethylamino-2-ethyl methacrylate (DMAEMA). The obtained block copolymers were characterized by gel permeation chromatography (GPC), $^1H$ NMR and FTIR-IR. The critical micelle concentration (CMC) of copolymer was determined by fluorescence spectra, it can be found that with hydrophilic block (PDMAEMA) increasing, CMC value of the polymer sample increased accordingly, and the CMC value was 0.012 mg/mL, 0.025 mg/mL and 0.037 mg/mL for $PCL_{50}$-b-$PDMAEMA_{68}$, $PCL_{50}$-b-$PDMAEMA_{89}$, $PCL_{50}$-b-$PDMAEMA_{112}$, $PCL_{50}$-b-$PDMAEMA_{89}$ was chosen as drug carrier to study in vitro release profile of anti-cancer drug (taxol). The temperature and pH dependence of the values of hydrodynamic diameter (Dh) of micelles, and self-assembly of the resulting block copolymers in water were evaluated by dynamic light scattering (DLS). The result showed that with the temperature increasing and pH decreasing, the Dh decreased. Drug-loaded nanoparticles were fabricated using paclitaxel as model. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) had been explored to study the morphology of the hollow micelles and the nanoparticles, revealing well-dispersed spheres with the average diameters both around 80 nm. In vitro release kinetics of paclitaxel from the nanoparticles was also investigated in different conditions (pH and temperature, etc.), revealing that the drug release was triggered by temperature changes upon the lower critical solution temperature (LCST) at pH 7.4, and at $37^{\circ}C$ by an increase of pH.

• 천문학회지
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• 제35권4호
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• pp.175-185
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• 2002

Almost half of primeval galaxies show P-Cygni type profiles in the Ly$\alpha$ emission line. The main underlying mechanism for the profile formation in these systems is thought to be the frequency re-distribution of the line photons in expanding scattering media surrounding the emission source. A Monte Carlo code is developed to investigate the Ly$\alpha$ line transfer in an optically thick and moving medium with a careful consideration of the scattering in the damping wings. Typical column densities and expansion velocities of neutral hydrogen investigated in this study are $N_{H1}{\~}10^{17-20}\;cm^{-2}$ and ${\Delta}V{\~} 100 km\;s^{-1}$. We investigate the dependence of the emergent profiles on the kinematics and on the column density. Our numerical results are applied to show that the damped Ly$\alpha$ absorbers may possess an expanding H I supershell with bulk flow of ${\~}200 km\;s^{-l}$ and H I column density $N_{H1}{\~}10^{19}\;cm^{-2}$. We briefly discuss the observational implications.