• BMB Reports
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• 제30권5호
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• pp.356-361
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• 1997

We cloned a cDNA (pPRC-1) which was comprised of 841 nucleotides from the cDNA library of a male ICR mouse submandibular gland ($SMG^+$). The nucleotide sequences of pPRC-1 were identical to those of exons 2 and 3 of the mGK-21 gene, a potentially functional glandular kallikrein identified in a Balb/c mouse, except for one nucleotide residue. Although this substitution changes Ile (ATT) in pPRC-1 to Val (GTT) in mGK-21, this difference has been explained by strain polymorphism. From the amino acid sequences predicted from its cDNA, we speculated that mGK-21 gene products/pGK21 consist of 261 amino acids including the $NH_2$-terminal signal peptide (residues 1~17), the short propeptide (residues 17~24), and the active peptide (residues 25~261). Although we did not demonstrate the enzyme activity of pGK21, it was assumed that pGK 21 was involved in the maturation of certain bioactive polypeptide(s) in mouse SMG for the following reasons : (a) mGK-21 gene was apparently expressed in a male ICR mouse SMG: (b) the proposed active site $His^{65}$, $Asp^{120}$, and $Ser^{213}$ residues were completely conserved in pGK21 just like other glandular kallikreins; (c) the cloned cDNA was translated to a predicted 27 kDa polypeptide chain in vitro: (d) the 27 kDa polypeptide chain produced by CHO cells was produced to a putative active form by trypsin.

• Journal of Microbiology
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• 제39권1호
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• pp.37-41
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• 2001

The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

• BMB Reports
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• 제30권2호
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• pp.113-118
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• 1997

Aspartase from Hafnia alvei was inactivated by N-ethylmaleimide (NEM) and 5,5' -Dithiobis-(2-znitrobenzoic acid) (DTNB) following pseudo-first order kinetics. Their apparent reaction orders were 0.83 and 0.50 for NEM and DTNB modifications, respectively, indicating that inactivation was due to a sulfhydryl group in the active site of aspartase and participation of the sulfhydryl group in an essential step in the catalytic reaction. When aspartase was modified by DTNB, the enzyme activity was restored by dithiothreitol treatment, indicating that cysteine residuetsl islarel possibly at or near the active site. The pH-dependence of the inactivation rate by NEM suggested that an amino acid residue having pK value of 8.3 was involved in the inactivation. When aspartase was incubated with NEM and L-aspartate together, L-aspartate markedly protected the enzyme from inactivation by NEM, but the other reagents used did not.

• 통합자연과학논문집
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• 제8권4호
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• pp.267-272
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• 2015

Galactosemia is a potentially lethal disorder caused by the deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT) within the Leloir pathway. Galactokinase (GALK) is the enzyme in Leloir pathway which converts ${\alpha}$-D galactose to galactose 1-phosphate. The elevated level of galactose-1-phosphate, the product of GALK plays a major role in Galactosemia. Therefore the inhibition of GALK is a novel therapy for this disorder. Hence in the present study, we performed molecular docking of twenty inhibitors with different activity against galactokinase into the active site of galactokinase enzyme. The binding mode of these inhibitors was obtained using Surflex dock program interfaced in Sybyl-X2.0. The residues such as SER141, TYR109, ARG105, ARG228, TYR106, GLY346, GLY136, ASP86, ASP186 and SER142 found to interact with inhibitors.

• BMB Reports
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• 제30권4호
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• pp.280-284
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• 1997

Phenylglyoxal diethyl pyrocarbonate (DEPC), and 1-cyclohexyl-3-[2-morpholinoethyl]-carbodiimide metho-p-toluenesulfonate (CMC) are modifying reagents specific for arginine, histidine, and aspartate or glutamate, respectively. They were found to inactivate S. cerevisiae farnesyl protein transferase (FPTase). The peptide substrate protected the enzyme against inactivation by CMC and the other substrate farnesyl pyrophosphate showed protection against inactivation by phenylglyoxal. while neither of the two substrates protected the enzyme against DEPC inactivation. These results suggest the presence of aspartate/glutamate, arginine and histidine residues at the active site of this enzyme.

• BMB Reports
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• 제31권2호
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• pp.139-143
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• 1998

The catabolic ${\alpha}-acetolactate$ synthase purified from Serratia marcescens ATCC 25419 was rapidly inactivated by the tryptophane-specific reagent, N -bromosuccinimide, and the arginine-specific reagent, phenylglyoxal. The enzyme was inactivated slowly by the cysteine-specific reagent N-ethylmaleimide. The second-order rate constants for the inactivation by N-bromosuccinimide, phenylglyoxal. and N -ethylmaleimide were $114,749M^{-1}min^{-1}$, $304.3M^{-1}min^{-1}$, and $5.1M^{-1}min^{-1}$, respectively. The reaction order with respect to N-bromosuccinimide, phenylglyoxal, and N-ethylmaleimide were 1.5,0.71, and 0.86, respectively. The inactivation of the catabolic aacetolactate synthase by these modifying reagents was protected by pyruvate. These results suggest that essential tryptophane, arginine, and cysteine residues are located at or near the active site of the catabolic ${\alpha}-acetolactate$ synthase.

• Journal of Microbiology and Biotechnology
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• 제26권5호
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• pp.837-845
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• 2016

A novel endodextranase isolated from Paenibacillus sp. was found to produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides with a degree of polymerization of 7-14 from dextran. To determine the active site, the enzyme was modified with 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide (EDC) and α-epoxyalkyl α-glucosides (EAGs), an affinity labeling reagent. The inactivation followed pseudo first-order kinetics. Kinetic analysis and chemical modification using EDC and EAGs indicated that carboxyl groups are essential for the enzymatic activity. Three Asp and one Glu residues were identified as candidate catalytic amino acids, since these residues are completely conserved across the GH family of 66 enzymes. Replacement of Asp189, Asp340, or Glu412 completely abolished the enzyme activity, indicating that these residues are essential for catalytic activity.

• 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.

• BMB Reports
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• 제35권3호
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• pp.255-261
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• 2002

GTP cyclohydrolase I (E.C. 3.5.4.16) is a homodecameric protein that catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (H2NTP), the initial step in the biosynthesis of pteridines. It was proposed that the enzyme complex could be composed of a dimer of two pentamers, or a pentamer of tightly associated dimers; then the active site of the enzyme was located at the interface of three monomers (Nar et al. 1995a, b). Using mutant enzymes that were made by site-directed mutagenesis, we showed that a decamer of GTP cyclohydrolase I should be composed of a pentamer of five dimers, and that the active site is located between dimers, as analyzed by a series of size exclusion chromatography and the reconstitution experiment. We also show that the residues Lys 136, Arg139, and Glu152 are of particular importance for the oligomerization of the enzyme complex from five dimers to a decamer.

• Journal of Microbiology and Biotechnology
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• 제16권8호
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• pp.1325-1329
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• 2006

Structural modeling of $\beta$-galactosidase from L. lactis ssp. lactis 7962 has shown that the residues Cys-472 and His-509 are located in the wall of the active-site cavity. To examine the functions of Cys-472 and His-509, we generated five site-specific mutants: Cys-472-Ser, Cys-472-Thr, Cys-472-Met, His-509-Asn, and His-509-Phe. $\beta$-Galactosidase substituted at Cys-472 with Met or His-509 with Phe had <3% of the activity of the native enzyme when assayed using ONPG as substrate. The other mutants Cys-472-Ser, Cys-472-Thr, and His-509-Asn had ca. 10-15% of the native enzyme activity. The V$_max$ values of the five mutated enzymes were lower (60-7,000-fold) than that of native enzyme. These results show that the catalytic ability of $\beta$-galactosidase is significantly affected by mutations at Cys-472 or His-509.