• Bulletin of the Korean Chemical Society
• /
• v.27 no.10
• /
• pp.1618-1622
• /
• 2006

Bacteriophage T7 gp4A' is a ring-shaped hexameric DNA helicase that catalyzes duplex DNA unwinding using dTTP hydrolysis as an energy source. To investigate the effect of single-stranded DNA (ssDNA) on the kinetic pathway of dTTP hydrolysis by the T7 DNA helicase complexed with ssDNA, we have first determined optimal concentration of long circular M13 single-stranded DNA and pre-incubation time in the absence of $Mg^{2+}$ which is necessary for the helicase-ssDNA complex formation. Steady state dTTP hydrolysis in the absence of $Mg^{2+}$ by the helicase-ssDNA complex provided $k_{cat}$ of $8.5\;{\times}\;10^{-3}\;sec^{-1}$. Pre-steady state kinetics of the dTTP hydrolysis by the pre-assembled hexameric helicase was monitored by using the rapid chemical quench-flow technique both in the presence and absence of M13 ssDNA. Pre-steady state dTTP hydrolysis showed distinct burst kinetics in both cases, indicating that product release step is slower than dTTP hydrolysis step. Pre-steady state burst rates were similar both in the presence and absence of ssDNA, while steady state dTTP hydrolysis rate in the presence of ssDNA was much faster than in the absence of ssDNA. These results suggest that single-stranded DNA stimulates dTTP hydrolysis reaction by T7 helicase by enhancing the rate of product release step.

• BMB Reports
• /
• v.40 no.1
• /
• pp.100-106
• /
• 2007

The wound-inducible lipoxygenase obtained from maize is one of the nontraditional lipoxygenases that possess dual positional specificity. In this paper, we provide our results on the determination and comparison of the kinetic constants of the maize lipoxygenase, with or without detergents in the steady state, and characterization of the dependence of the kinetic lag phase or initial burst, on pH, substrate, and detergent in the pre-steady state of the lipoxygenase reaction. The oxidation of linoleic acid showed a typical lag phase in the pre-steady state of the lipoxygenase reaction at pH 7.5 in the presence of 0.25% Tween-20 detergent. The reciprocal correlation between the induction period and the enzyme level indicated that this lag phenomenon was attributable to the slow oxidative activation of Fe (II) to Fe (III) at the active site of the enzyme as observed in other lipoxygenase reactions. Contrary to the lagging phenomenon observed at pH 7.5 in the presence of Tween-20, a unique initial burst was observed at pH 6.2 in the absence of detergents. To our knowledge, the initial burst in the oxidation of linoleic acid at pH 6.2 is the first observation in the lipoxygenase reaction. Kinetic constants (Km and kcat values) were largely dependent on the presence of detergent. An inverse correlation of the initial burst period with enzyme levels and interpretations on kinetic constants suggested that the observed initial burst in the oxidation of linoleic acid could be due to the availability of free fatty acids as substrates for binding with the lipoxygenase enzyme.

• Animal cells and systems
• /
• v.8 no.4
• /
• pp.307-312
• /
• 2004

Fast kinetics of transient pH changes and difference spectrum formation have been investigated following mixing of ADP/ATP with partially purified plasma membrane PM-ATPase of the pathogenic yeast Candida albicans in the presence of five nutrients: glucose, glutamic acid, proline, lysine, and arginine and two analogs of glucose: 2-deoxy D-glucose and xylose. Average $H^+$- absorption to release ratio, indicative of population of ATPase undergoing complete hydrolytic cycle, was found to be 0.27 for control. This ratio varied between 0.25 (proline) to 0.36 (arginine) for all other compounds tested, except for glucose. In the presence of glucose, $H^+$- absorption to release ratio was exceptionally high (0.92). While no UV difference spectrum was observed with ADP, mixing of ATP with ATPase led to a large conformational change. Exposure to different nutrients restricted the magnitude of the conformational change; the analogs of glucose were found to be ineffective. This suppression was maximal in the case of glucose (80%); with other nutrients, the magnitude of suppression ranged from 40-50%. Rate of $H^+$- absorption, which is indicative of E~P complex dissociation, showed positive correlation with suppression of conformational change only in the case of glucose and no other nutrient/analog. Mode of interaction of glucose with plasma membrane $H^+$-ATPase thus appears to be strikingly distinct compared to that of other nutrients/analogs tested. The results obtained lead us to propose a model for explaining glucose stimulation of plasma membrane $H^+$-ATPase activity.