• BMB Reports
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• v.28 no.2
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• pp.112-117
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• 1995

Succinic semialdehyde reductase was inactivated by o-phthalaldehyde. The inactivation followed pseudo-first order kinetics, and the second-order rate constant for the inactivation process was 28 $M^{-1}s^{-1}$ at pH 7.4 and $25^{\circ}C$. The absorption spectrum ($\lambda_{max}$ 337 nm) and fluorescence excitation ($\lambda_{max}$ 340 nm) and fluorescence emission spectra ($\lambda_{max}$ 409 nm) were consistent with the formation of an isoindole derivative in the catalytic site between a cysteine and a lysine residue approximately about 3 $\AA$ apart. The substrate, succinic semialdehyde, did not protect enzymatic activity against inactivation, whereas the coenzyme NADPH protected against o-phthaladehyde induced inactivation of the enzyme. About 1 isoindole group per mol of the enzyme was formed following complete loss of enzymatic activity. These results suggest that the amino acid residues of the enzyme participating in a reaction with o-phthalaldehyde are cysteinyl and lysyl residues at or near the NADPH binding site.

• BMB Reports
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• v.30 no.5
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• pp.370-377
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• 1997

A novel assay method is described for rapid quantitation of reaction rate of sterol ${\Delta}^7$-reductase (${\Delta}^7$-SR) which catalyzes reduction of the ${\Delta}^7$-double bond of sterols. Of six different organ tissues-liver, small intestine, brain, lung, kidney, and testis-. ${\Delta}^7$-SR activity was detected only in liver (2.30 nmol/min/mg protein) and testis (0.11 nmol/min/mg protein). Using a newly developed method which employs diet-induced enzyme proteins and ergosterol as substrate, we assessed both kinetics ($K_m=210\;{\mu}M$, $V_{max}=1.93\;nmol/min/mg$) and inhibition of the rat hepatic ${\Delta}^7$-SR against well-studied cholesterol lowering agents such as triparanol ($IC_{50}=16\;{\mu}M$). 3-$\beta$-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A) ($IC_{50}=5.2\;{\mu}M$), and trans-1.4-bis(2-chlorobenzylaminomethyl)cyclohexane dihydrochloride (AY-9944) ($IC_{50}=0.25\;{\mu}M$). Of the three well-known AY-9944-sensitive cholesterogenic enzymes (i.e., ${\Delta}^7$-SR, sterol ${\Delta}^8$-isomerase, and sterol ${\Delta}^14$-reductase). ${\Delta}^7$-SR was found to be the most sensitive enzyme with a noncompetitive inhibition of this compound ($K_i=0.109\;{\mu}M$). Substrate specificity studies of the microsomal ${\Delta}^7$-SR indicate that the relative reaction rate for 7-dehydrocholesterol and ergosterol are 5.6-fold and 1.6-fold higher than that for lathosterol. ${\Delta}^7$-SR activity was also modulated by feeding rats a diet supplemented with 0.5% ergosterol (>2.6-fold) in addition to 5.0% cholestyramine plus 0.1% lovastatin ($\simeq$5.0-fold). Finally, microsomal ${\Delta}^7$-SR was solubilized by 1.5% 3-[3-(cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and enriched on PEG (0~10%) precipitation, which should be suitable for further purification of the enzyme.

• BMB Reports
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• v.42 no.1
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• pp.47-52
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• 2009

Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent $K_m$ of 3 mM for L-alanine, and a $V_{max}$ of $295\;{\mu}moles/min/mg$, with the optimum activity occurring at $37^{\circ}C$ and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5'-phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a $K_i$of $160\;{\mu}M$ and 30 mM, respectively.

• BMB Reports
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• v.31 no.1
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• pp.25-30
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• 1998

Kinetic studies of L-Alanine dehydrogenase from Bacillus subtilis-catalyzed reactions in the presence of $Zn^{2+}$ were carried out. The substrate (L-alanine) saturation curve is hyperbolic in the absence of the metal ion but it becomes sigmoidal when $Zn^{2+}$ is added to the reaction mixture indicating the positive cooperative binding of the substrate in the presence of zinc ion. The cooperativity of substrate binding depends on the xinc ion concentration: the Hill coefficients ($n_H$) varied from 1.0 to 1.95 when the zinc ion concentration varied from 0 to $60\;{\mu}m$. The inhibition of AlaDH by $Zn^{2+}$ is reversible and noncompetitive with respect to $NAD^+$ ($K_i\;=\;5.28{\times}10^{-5}\;M$). $Zn^{2+}$ itself binds to AlaDH with positive cooperativity and the cooperativity is independent of substrate concentration. The Hill coefficients of substrate biding in the presence of $Zn^{2+}$ are not affected by the enzyme concentration indicating that $Zn^{2+}$ binding does not change the polymerization-depolymerization equilibria of the enzyme. Among other metal ions, $Zn^{2+}$ appears to be a specific reversible inhibitor inducing conformational change through the intersubunit interaction. These results indicate that $Zn^{2+}$ is an allosteric competitive inhibitor and substrate being a non-cooperative per se, excludes the $Zn^{2+}$ from its binding site and thus exhibits positive cooperativity. The allosteric mechanism of AlaDh from Bacillus subtilis is consistent with both MWC and Koshland's allosteric model.

• Applied Biological Chemistry
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• v.41 no.5
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• pp.355-362
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• 1998

The minor form of phosphofructokinase (EC 2.7.1.11; PFK), which was suggested to be of plastid origin from the host fraction of chickpea nodules, was isolated as a small protein with apparent molecular mass near 220 kDa and purified to a high degree. SDS-PAGE and western blot indicated that the enzyme was made up of a homotetrameric structure (55 kDa). The enzyme had sharp pH profiles with maximal activities at pH 8 and displayed Michaelis-Menten kinetics with respect to Fru-6-P and nucleoside triphosphate substrate at the pH optimum (pH 8) and at pH 7. MgATP was the most effective phosphoryl donor. Phosphoenolpyruvate was a potent inhibitor of minor PFK activity, and the enzyme was also strongly inhibited by 3-phosphoglycerate, 2-phosphoglycerate, and to a lesser extent, PPi. Minor PFK was weakly activated by KCl, NaCl and Pi, and was inhibitory at high concentration of KCl and Pi.

• Journal of Microbiology and Biotechnology
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• v.17 no.5
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• pp.761-768
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• 2007

Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHI fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene(inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.

• Journal of Life Science
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• v.23 no.11
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• pp.1365-1370
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• 2013

Immobilized cellulase on weak ion exchange resin showed a typical Langmuir adsorption isotherm. Immobilized cellulase had better stability with respect to pH and temperature than free cellulase. Kinetics of thermal inactivation on free and immobilized cellulase followed first order rate, and immobilized cellulase had a longer half-life than free cellulase. The initial rate method was used to characterize the kinetic parameters of free and immobilized enzyme. The Michaelis-Menten constant $K_m$ was higher for the immobilized enzyme than it was for the free enzyme. The effect of the recirculation rate on cellulose degradation was studied in a recycling packed-bed reactor. In a continuous packed-bed reactor, the increasing flow rate of cellulose decreased the conversion efficiency of cellulose at different input lactose concentrations. Continuous operation for five days was conducted to investigate the stability of long term operation. The retained activity of the immobilized enzymes was 48% after seven days of operation.

• Korean Journal of Food Science and Technology
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• v.32 no.5
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• pp.1158-1167
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• 2000

To produce ${\beta}-cyclodextrin({\beta}-CD)$, a cyclodextrin glucanotransferase(CGTase) producing Aspergillus sp. CC-2-1 was isolated from soil. The enzyme was purified and its enzymological characteristics were investigated. It was found that production of CGTase reached to the maximum when the wheat bran medium containing 0.1% albumin, 2% $(NH_4)_2S_2O_8$, 2% soluble starch and 0.2% $KH_2PO_4$ was cultured for 5 days at $37^{\circ}C$. The purity of CGTase was increased by 13.14 folds after DEAE-cellulose ion exchange chromatography and Sephadex G-100, G-150 gel filtration and the specific activity was 172.14 unit/mg. Purified enzyme was confirmed as a single band by the polyacrylamide gel electrophoresis. The molecular weight of CGTase was estimated to be 27,800 by Sephadex G-100 gel filtration and SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature for the CGTase activity were 9.0 and $80^{\circ}C$, respectively. The enzyme was stable in pH $8.0{\sim}11.0$ at $60{\sim}80^{\circ}C$. The activity of purified enzyme was activated by $K^+,\;Cu^{2+}$ and $Zn^{2+}$. The activity of the CGTase was inhibited by the treatment with 2,4-dinitrophenol and iodine. The result suggests that the purified enzyme has phenolic hydroxyl group of tyrosine, histidine imidazole group and terminal amino group at active site. The reaction of this enzyme followed typical Michaelis-Menten kinetics with the $K_m$ value of 18.182 g/L with the $V_{max}$ of 188.68 ${\mu}mole/min$. The activation energy for the CGTase was calculated by Arrhenius equation was 1.548 kcal/mol.

• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.1897-1903
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• 2006

In order to investigate the functions of the C-terminal region of chitinase ChiCW of Bacillus cereus 28-9, a C-terminal truncated enzyme, ChiCW$\Delta$FC, was expressed in Escherichia coli and purified to homogeneity for biochemical characterization. Compared with ChiCW, ChiCW$\Delta$FC exhibited higher chitinase activity at high temperature and pH, but expressed lower hydrolytic and binding activities toward insoluble substrates. In addition, kinetic properties indicated that ChiCW$\Delta$MC hydrolyzed oligomeric and polymeric substrates less efficiently than ChiCW. These results suggest that the C-terminal region of ChiCW plays important roles in substrate binding and hydrolysis of chitin. In addition, the biological meaning of C-terminal proteolytic modification of ChiCW is discussed.

• Microbiology and Biotechnology Letters
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• v.19 no.6
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• pp.610-613
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• 1991

Some of the Kinetic properties of crystallic xanthine dehydrogenase form Pseudomonas synxantha A3 were studied. The enzyme activity was strongly inhibited by adenine, 8-azaadenine, 2-methyladenine, guanine, and 8-azaguanine, but not by caffeine, and the inhibitions by adenine and guanine were observed to be of noncompetitive type. The $K_i$ values for adenine and guanine were 0.037 and 0.098 mM, respectively. Michaelis constants were found to be 0.33 and 0.06 rnM for hypoxanthine and xanthine with $NAD^+$ as the second substrate, respectively, and 0.1 rnM for $NAD^+$ with either hypoxanthine or xanthine as the second substrate.