• Journal of Microbiology
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• v.39 no.1
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• pp.24-30
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• 2001

In addition to effecting the catalysis of sugar uptake, the bacterial phosphoenolpyruvate::sugar phosphotransferase system regulates a variety of physiological processes. In a previous paper [Seok et al.,(1997) J. Biol. Chem. 272, 26511-26521], we reported the interaction with and allosteric regulation of Esiherichia coli glycogen phosphorylase activity by the histidine-containing phosphocarrier protein HPr in vitro. Here, we show that the specific interaction between HPr and glycogen phosphorylase occurs in vivo. To address the physiological role of the HPr-glycogen phosphorylase complex, intracellular glycogen levels were measured in E. coli strains transformed with various plasmids. While glycogen accumulated during the transition between exponential and stationary growth phases in wildtype cells, it did not accumulate in cells overproducing HPr or its inactive mutant regardless of the growth stage. From these results, we conclude that HPr mediates crosstalk between sugar uptake through the phosphoenolpyruvate:sugar phosphotransferase system and glycogen breakdown. The evolutionary significance of the HPr-glycogen phosphorylase complex is suggested.

• 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.

• BMB Reports
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• v.40 no.6
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• pp.1077-1082
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• 2007

Human glutamate dehydrogenase exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. In the nervous system, GDH is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter during neurotransmission. Chloroquine has been known to be a potent inhibitor of house-keeping GDH1 in permeabilized liver and kidneycortex of rabbit. However, the effects of chloroquine on nerve-specific GDH2 have not been reported yet. In the present study, we have investigated the effects of chloroquine on hGDH2 at various conditions and showed that chloroquine could inhibit the activity of hGDH2 at dose-dependent manner. Studies of the chloroquine inhibition on enzyme activity revealed that hGDH2 was relatively less sensitive to chloroquine inhibition than house-keeping hGDH1. Incubation of hGDH2 was uncompetitive with respect of NADH and non-competitive with respect of 2-oxoglutarate. The inhibitory effect of chloroquine on hGDH2 was abolished, although in part, by the presence of ADP and L-leucine, whereas GTP did not change the sensitivity to chloroquine inhibition. Our results show a possibility that chloroquine may be used in regulating GDH activity and subsequently glutamate concentration in the central nervous system.

• Biomolecules & Therapeutics
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• v.20 no.2
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• pp.133-143
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• 2012

Matrix metalloproteinases (MMPs) are a subfamily of zinc-dependent proteases that are re-sponsible for degradation and remodeling of extracellular matrix proteins. The activity of MMPs is tightly regulated at several levels including cleavage of prodomain, allosteric activation, com-partmentalization and complex formation with tissue inhibitor of metalloproteinases (TIMPs). In the central nervous system (CNS), MMPs play a wide variety of roles ranging from brain devel-opment, synaptic plasticity and repair after injury to the pathogenesis of various brain disorders. Following general discussion on the domain structure and the regulation of activity of MMPs, we emphasize their implication in various brain disorder conditions such as Alzheimer's disease, multiple sclerosis, ischemia/reperfusion and Parkinson's disease. We further highlight accumu-lating evidence that MMPs might be the culprit in Parkinson's disease (PD). Among them, MMP-3 appears to be involved in a range of pathogenesis processes in PD including neuroinflamma-tion, apoptosis and degradation of ${\alpha}$-synuclein and DJ-1. MMP inhibitors could represent poten-tial novel therapeutic strategies for treatments of neurodegenerative diseases.

• Molecules and Cells
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• v.19 no.1
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• pp.97-103
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• 2005

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the $K_m$ of the enzyme for NADH and ${\alpha}-ketoglutarate$ increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency ($k_{cat}/K_m$) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in $k_{cat}$ values. There was a linear relationship between incorporation of [$^{14}C$]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [$^{14}C$] incorporated per mol of monomer for wild type hGDHs. No incorporation of [$^{14}C$]p-chloromercuribenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.