• Bulletin of the Korean Chemical Society
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• 제10권1호
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• pp.107-111
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• 1989

Glucoamylase was inactivated with 1-ethyl-2-(dimethylaminopropyl)carbodiimide (EDC) at pH 5.0. Time course of inactivation of glucoamylase was at least biphasic. From the results of the titration of SH groups with Ellman's reagent and hydroxylamine treatment at pH 7.0, it was concluded that the crucial sites of modification were carboxyl groups of glucoamylase. The CD spectrum of EDC-modified glucoamylase suggested that the gross conformation of the native enzyme was retained. The inactivation of glucoamylase was reduced remarkably in the presence of maltose. The logarithm of the half-life of the inactivation of glucoamylase by EDC was a linear function of log[EDC] in each stage indicating that one carboxyl group among the modified ones was crucial for inactivation of glucoamylase. The change in the binding affinity due to modification was determined by using an affinity column. It indicates that the carboxyl group of glucoamylase seems to play a role in both, the catalysis and substrate binding in the first stage, but in the second stage the binding affinity is recovered almost up to that of native enzyme.

• Archives of Pharmacal Research
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• 제22권5호
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• pp.464-473
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• 1999

Regulation of phosphcholine-hydrolyzing phosphatase (phosphocholine-phosphatase) activity, purified from bovine brain, was examined under physiological conditions. Various endogenous phosphomonoesters, which were utilized as substrate, inhibited the phosphocoline-phosphatase activity competitively (Ki 5.5-$82.0 {\mu}M$); among phosphomonoesters tested, there was a similar order of capability between the binding affinity of substrate and the inhibitory potency. In addition, phosphate ions also inhibited the phosphatase activity competitively with a Ki value of approximately $16{\mu}M$. Although leucine or theophylline inhibited the phosphatase activity at pH 9.0, their inhibitory action decreased greatly at pH 7.4. The pH-Km and pH-Vm profiles indicate that ionizable amino acids are involved in substrate binding as well as catalysis, alluding that the phosphatase activity may be highly dependent on the intracellular pH. Amino acid modification study supports the existence of tyrosine, arginine or lysine residue in the active site, and the participation of tyrosine residue in the catalytic action may e suggested positively for the susceptibility to the action of tetranitromethane or HOl-generator. Separately, the oxidative inactivation of phosphocholine-phosphatase activity was investigated. Of oxidants tested, HOONO, HOCl, HOl and $ascorbate/Cu^{2+}$ system were effective to inactivate the phosphatase activity. Noteworthy, a remarkable inativation was accomplished by $30{\mu}M$ HOCl in combination with 1 mM Kl. Inaddition, $Cu^{2+}(3{\mu}M) $in combination with ascorbate at concentrations as low as 0.1-0.3 mM reduced the phosphatase activity to a great extent. From these results, it is proposed that the phosphocholine-phosphatase activity may be regulated endogenously and susceptible to the various oxidant system in vivo.

• Journal of Microbiology and Biotechnology
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• 제22권12호
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• pp.1724-1730
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• 2012

An ${\alpha}$-L-arabinofuranosidase (TmAFase) from Thermotoga maritima MSB8 is a highly thermostable exo-acting hemicellulase that exhibits a relatively higher activity towards arabinan and arabinoxylan, compared with other glycoside hydrolase 51 family enzymes. In the present study, we carried out the enzymatic characterization and structural analysis of TmAFase. Tight domain associations found in TmAFase, such as an inter-domain disulfide bond (Cys306 and Cys476) in each monomer, a novel extended arm (amino acids 374-385) at the dimer interface, and total 12 salt bridges in the hexamer, may account for the thermostability of the enzyme. One of the xylan binding determinants (Trp96) was identified in the active site, and a region of amino acids (374-385) protrudes out forming an obvious wall at the substrate-binding groove to generate a cavity. The altered cavity shape with a strong negative electrostatic distribution is likely related to the unique substrate preference of TmAFase towards branched polymeric substrates.

• 농약과학회지
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• 제9권3호
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• pp.191-198
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• 2005

신규 살충제 표적 단백질인 AnCE의 활성부위 잔기들과 상호작용 가능한 약리단 (pharmacophore)을 세 개의 펩타이드로 이루어진 ACE 기질 Hippuryl-L -histidyl-L-leucine (Hip-L-His-L-Leu, HHL) 분자의 구조를 모델로 하여 예측하였다. HHL의 분자구조, 용액장 내에서의 구조변화 그리고 약리단을 구성하는 원자들의 전하밀도 분석을 위해 순이론적인 양자화학 계산방법을 이용하여 구조 최적화, NMR 화학적 이동 및 NPA 계산을 수행하였다. 이론적인 NMR 화학적 이동 값들은 실험 결과와 잘 일치함을 보였고 전하밀도 계산 결과는 해당원자의 약리단을 분석하는데 사용되었다. 결과적으로 HHL 분자 구조를 통해 소수성(aromatic, aliphatic), 수소결합 주게, 수소결합 받게, 금속 아연 결합부위의 5개 약리단을 추출할 수 있었다.

• Journal of Microbiology and Biotechnology
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• 제28권4호
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• pp.571-578
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• 2018

Biofuel production using lignocellulosic biomass is gaining attention because it can be substituted for fossil fuels without competing with edible resources. However, because Saccharomyces cerevisiae does not have a ${\text\tiny{D}}$-xylose metabolic pathway, oxidoreductase or isomerase pathways must be introduced to utilize ${\text\tiny{D}}$-xylose from lignocellulosic biomass in S. cerevisiae. To elucidate the biochemical properties of xylose isomerase (XI) from Piromyces sp. E2 (PsXI), we determine its crystal structure in complex with substrate mimic glycerol. An amino-acid sequence comparison with other reported XIs and relative activity measurements using five kinds of divalent metal ions confirmed that PsXI belongs to class II XIs. Moreover kinetic analysis of PsXI was also performed using $Mn^{2+}$, the preferred divalent metal ion for PsXI. In addition, the substrate-binding mode of PsXI could be predicted with the substrate mimic glycerol bound to the active site. These studies may provide structural information to enhance ${\text\tiny{D}}$-xylose utilization for biofuel production.

• Bulletin of the Korean Chemical Society
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• 제20권3호
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• pp.301-306
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• 1999

Hepatitis C virus (HCV) has been known to be an enveloped virus with a positive strand RNA genome and the major agent of the vast majority of transfusion associated cases of hepatitis. For viral replication, HCV structural proteins are first processed by host cell signal peptidases and NS2/NS3 site of the nonstructural protein is cleaved by a zinc-dependent protease NS2 with N-terminal NS3. The four remaining junctions are cleaved by a separate NS3 protease. The solution conformations of NS4B/5A, NS5A/5B substrates and NS5A/5B inhibitor have been determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. NMR data suggested that the both NS5A/5B substrate and inhibitor appeared to have a folded tum-like conformation not only between P1 and P6 position but also C-terminal region, whereas the NS4B/5A substrate exhibited mostly extended conformation. In addition, we have found that the conformation of the NS5A/5B inhibitor slightly differs from that of NS5A/5B substrate peptide, suggesting different binding mode for NS3 protease. These findings will be of importance for designing efficient inhibitor to suppress HCV processing.

• 통합자연과학논문집
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• 제4권4호
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• pp.273-277
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• 2011

It's a threat for the public health that H1N1 (Influenza virus A) causes disease and transmits among humans. WHO (world health organization) declared that the infections caused by the new strain had reached pandemic proportions. The approved neuraminidase inhibitors (Zanamivir and Oseltamivir) and related investigative drug (BCX-1812) are potent, specific inhibitors of influenza A and B viruses. These drugs are highly effective to prevent influenza A and B infections. Early therapeutic use reduces illness duration and respiratory complications. Recently, we found one of the potent inhibitor of erystagallin A ($IC_{50}$ of 2.04 ${\mu}M$) for neuraminidase target, this inhibitor shows most similar structure to its natural substrate, sialic acid. Therefore, we chose 1l7f to get the receptor structure for docking study among many crystal structures. A docking study has been performed in Surflex-Dock module in SYBYL 8.1. In the present study, we attempt to compare the docking studies of pterocarpin and erystagallin A with neuraminidase receptor structure. In the previous report, the methoxy group of pterocarpin had H-bonding with Arg residues. The present docking results for erystagallin A showed the backbone of hydroxyl group shows significant H-bonding interactions with Arg152 and Arg292. The results showed that erystagallin A interacts more favorably with distinctive binding site rather than original active site. Therefore, we tried to reveal plausible binding mode and important amino acid for this inhibitor using docking and site id search calculations of Sybyl. The results obtained from this work may be utilized to design novel inhibitors for neuraminidase.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1434-1440
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• 2006

The stable anchorage of pyridoxal 5'-phosphate (PLP) in the active site of D-amino acid transaminase (D-AT) is crucial for the enzyme catalysis. The three-dimensional structure of D-AT revealed that Glu32 is one of the active site groups that may playa role in PLP binding. To prove the role of Glu32 in PLP stability, we firstly checked the rate of the potential rate-limiting step. The kinetic analysis showed that the rate of the ${\alpha}$-deprotonation step reduced to 26-folds in E32A mutant enzyme. Spectral analyses of the reaction of D-AT with D-serine revealed that the E32A mutant enzyme failed to stabilize the key enzyme-substrate intermediate, namely a quinonoid intermediate (E-Q). Finally, analysis of circular dichroism (CD) on the wild-type and E32A mutant enzymes showed that the optical activity of PLP in the enzyme active site was lost by the removal of the carboxylic group, proving that Glu32 is indeed involved in the cofactor anchorage. The results suggested that the electrostatic interaction network through the groups from PLP, Glu32, His47, and Arg50, which was observed from the three-dimensional structure of the enzyme, plays a crucial role in the stable anchorage of the cofactor to give necessary torsion to the plane of the cofactor-substrate complex.

• BMB Reports
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• 제38권1호
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• pp.58-64
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• 2005

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.

• 미생물학회지
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• 제49권2호
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• pp.105-111
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• 2013

Erm 단백질은 23S rRNA의 특정 아데닌 잔기 $N_6$ 위치에 methylation을 일으켜 임상적으로 중요하게 사용되는 macrolide-lincosamide-streptogramin B계 항생제에 내성을 유발시킨다. 최근 ErmC'에서 N-말단 catalytic domain과 C-말단 substrate binding domain를 연결하는 오목한 홈 형성부위에 존재하는 잘 보존된 아미노산 잔기가 기질과 상호작용하는 것으로 제안되었다. 우리는 ErmSF에서 두 domain의 연결 부위의 오목한 홈에 위치하여 기질과의 상호작용이 예상되며 또한 Erm 단백질들 사이에서 매우 높게 보존되어있는 237번 아르지닌 잔기를 치환하여 그 기능을 in vivo, in vitro상에서 검색하여 분석하였다. R237A 변이 단백질을 발현하는 세균은 야생형 단백질을 발현하는 세균과 비교하여 in vivo 상에서는 차이를 나타내지 않았으나 순수분리 한 후 in vitro에서의 효소 활성은 야생형에 비하여 51%만을 나타내어 그 잔기가 기질 부착 기능을 수행하고 있다고 제안할 수 있었다.