• Bulletin of the Korean Chemical Society
• /
• v.30 no.8
• /
• pp.1724-1728
• /
• 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 $Mg^{2+}$.

• Journal of Microbiology
• /
• v.37 no.2
• /
• pp.59-65
• /
• 1999

RecA protein can promote strand assimilation, homologous pairing, and strand exchange. All these reactions require DNA-dependent ATP hydrolysis by recA protein, and the activities of recA protein are affected by the ionic environment. In this experiment, DNA-dependent ATPase activity showed different sensitivity to anionic species. ATP hydrolysis and strand exchange were relatively sensitive to salt in the reactions with NaCl, strongly inhibited at 100 mM NaCl. However, the inhibition by sodium acetate or sodium glutamate was not observed at 50∼100 mM concentration. Addition of sodium glutamate to the standard reaction condition increased the apparent efficiency of ATP hydrolysis during strand exchange. The condition including 50∼100 mM sodium-glutamate might be similar to the physiological condition.

• Proceedings of the PSK Conference
• /
• 2002.10a
• /
• pp.336.1-336.1
• /
• 2002

HSP100/Clp family functions as molecular chaperone and ATP dependent protease. The Streptococcus pneumoniae ClpL. a homologue of bacterial ClpB and yeast cytosolic HSP 104. is one of major heat shock proteins but its biochemical properties are unknown. In this study. ClpL in Streptococcus pneumoniaewas characterized using histidine tagged recombinant ClpL. When ATP hydrolysis activity was compared in the presence or absence of a variety of nucleotides or divalent ions. either ATP or Mn$2^＋$ ion was found to increase significantly the rate of ATP hydrolysis. Furthermore. glutaraldehyde cross-linking and subsequent native-PAGE analfysis showed that ClpL forms dimer. but in the presence of 4 mM concentration of $Mn^{2＋}$ion as a cofactor for ATP hydrolysis and oligormerization in vitro.

• Journal of Photoscience
• /
• v.2 no.1
• /
• pp.27-29
• /
• 1995

Site directed mutagenesis has been introduced to determine active site(s) and molecular structure of E. coli RecA protein. Recombinant DNAs were constructed by point mutation of Tyr-264 to Phe which assumed active site for binding and hydrolysis of ATP. RecA proteins were purified from recombinants containing wild type and mutant genes and analyzed for ATPase activity assay. Result suggests that Tyr-264 is involved in ATP binding rather than ATP hydrolysis.

• Korean Journal of Microbiology
• /
• v.46 no.4
• /
• pp.313-318
• /
• 2010

Deinococcus radiodurans RecA protein, when bound to DNA, exhibits a DNA-dependent ATPase. In the absence of DNA, the rate of RecA protein-promoted ATP hydrolysis drops 1,000-fold under the physiological concentrations of salt. This DNA-independent activity can be stimulated to levels approximating those observed with DNA by adding high concentrations (approximately 1.6 M) of a wide variety of salts. This effect was characterized by varying salt concentration and comparing the effects of different ion types. The higher concentrations of salt stimulated the ATP hydrolysis by RecA protein in the absence of DNA. At 1.6 M chloride, the observed stimulation showed the following cation trend $K^+{\geq}Na^+$ > $NH_4^+$ and the following anion sequence was observed: $glutamate^- \; > \; C1^- \;> \; acetate^-\; > \;PO_4^-$ at 1.6 M $K^+$. The catalytic properties of the salt-stimulated ATP hydrolysis reaction was optimal between pH 7.0 and 8.0, which was similar to the double stran nded DNA-dependent ATPase activities of Deinococcus radiodurans RecA protein. In the absence of DNA the active species for ATP hydrolysis by RecA protein was shown to be an aggregate of three RecA protein molecules.

• Microbiology and Biotechnology Letters
• /
• v.27 no.5
• /
• pp.354-358
• /
• 1999

In order to know the antifungal characteristics of saturated fatty acids having 6 to 12 carbons, their minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) were estimated against Saccharomyces cerevisiae. Fatty acids from C6 to C11 exhibited increasing activity with chain length, but C12 fatty acid did not show activity at all. In relation to antifungal modes of actions, fatty acids investigated showed on inhibitory activity toward the plasma membrane H+-ATPase of Saccharomyces cerevisiae. Their inhibitions to the glucose-induced acidification and ATP hydrolysis caused by the proton pump were found to be in common wiht antifungal activities. At the test concentration of 1mM, hexanoic acid (C6) showed the lowest inhibition of about 30%, while undecanoic acid(C11) showed the strongest inhibition of over 90%. In addition, as seen with antifungal activity, the inhibitory activity of dodecanoic acid (C12) was suddenly reduced to less than 50%.

• Microbiology and Biotechnology Letters
• /
• v.21 no.1
• /
• pp.30-35
• /
• 1993

Clp is a relatively abundant ATP-dependent protease found in E. coli. Its specific activity was proportional to the concentration of the limiting amount of Clp A and an excess amount of Clp P, and vice versa. Clp A has an intrinsic ATPase activity that is stimulated by casein, and contains a second site for binding A TP, in addition to the ATPase site. The modification of sulfhydryl groups in Clp A with reagents which have bulky groups such as N-phenylmaleimide led to nullifying both ATPase and protease activity. The same sites were modified by sulfhydryl reagents. It seems that the sulfhydryl groups of Clp A are not directly involved in catalysis. Since non-hydrolyzable analogs of ATP do not activate Clp, ATP hydrolysis may be essential for the proteolytic activity of Clp protease. Clp A and Clp P did not associate in the absence of nucleotide. The results suggest that the activity of the proteolytic component, Clp P, is regulated by the A TP-dependent cycling of Clp A between the activator form and the non-activator form.

• Korean Journal of Microbiology
• /
• v.43 no.4
• /
• pp.250-255
• /
• 2007

The RecA protein of Deinococcus radiodurans is essential for the extreme radiation resistance of this organism. The central steps involved in recombinational DNA repair require DNA-dependent ATP hydrolysis by recA protein. Key feature of RecA protein-mediated activities is the interactions with ssDNA and dsDNA. The ssDNA is the site where RecA protein filament formation nucleates and where initiation of DNA strand exchange takes place. The effect of sequence heterogeneity of ssDNA was examined in this experiment. The rate of homopolymeric synthetic ssDNA-dependent ATP hydrolysis was constant or nearly so over a broader range of pHs. For poly(dT)-dependent ATP or dATP hydrolysis, rates were generally faster, with a broader optimum between pH 7.0 and 8.0. Activities of RecA protein were affected by the ionic environment. The ATPase activity was shown to have different sensitivity to anionic species. The presence of glutamate seemed to slimulate the hydrolytic activity. Dr RecA protein was shown to require $Mg^{2+}$ ion greater than 2 mM for binding to etheno ssDNA and the binding stoichiometry of 3 nucleotide for RecA protein monomer.

• Molecules and Cells
• /
• v.44 no.2
• /
• pp.79-87
• /
• 2021

Chromatin dynamics is essential for maintaining genomic integrity and regulating gene expression. Conserved bromodomain-containing AAA+ ATPases play important roles in nucleosome organization as histone chaperones. Recently, the high-resolution cryo-electron microscopy structures of Schizosaccharomyces pombe Abo1 revealed that it forms a hexameric ring and undergoes a conformational change upon ATP hydrolysis. In addition, single-molecule imaging demonstrated that Abo1 loads H3-H4 histones onto DNA in an ATP hydrolysis-dependent manner. However, the molecular mechanism by which Abo1 loads histones remains unknown. Here, we investigated the details concerning Abo1-mediated histone loading onto DNA and the Abo1-DNA interaction using single-molecule imaging techniques and biochemical assays. We show that Abo1 does not load H2A-H2B histones. Interestingly, Abo1 deposits multiple copies of H3-H4 histones as the DNA length increases and requires at least 80 bp DNA. Unexpectedly, Abo1 weakly binds DNA regardless of ATP, and neither histone nor DNA stimulates the ATP hydrolysis activity of Abo1. Based on our results, we propose an allosteric communication model in which the ATP hydrolysis of Abo1 changes the configuration of histones to facilitate their deposition onto DNA.

• Bulletin of the Korean Chemical Society
• /
• v.28 no.12
• /
• pp.2261-2265
• /
• 2007

Prevention of thermal aggregation of the denatured protein by the group II chaperonin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1 (ApcpnA) has been investigated. ApcpnA exists as a homo-oligomer in a ring structure, which protects thermal aggregation of the chemically denatured bovine rhodanese at 50 oC. ApcpnA alone is not sufficient for chaperonin activity, but the chaperonin activity is greatly enhanced in the presence of manganese ion and ATP. Compared to the mesophilic chaperonin GroEL/GroES, ApcpnA is more activated at a higher temperature and protects the aggregation-prone unfolded state of the denatured rhodanese from thermal aggregation. Binding of ATP is sufficient for ApcpnA to perform the chaperonin function in vitro, but hydrolysis of ATP is not necessarily required. We propose that utilization of Mn2+ and adenosine nucleotide regardless of ATP hydrolysis may be one of peculiar properties of archaeal chaperonins.