Fluorescence and kinetic studies on the divalent metal ion induced conformational changes in DNase a.
DNase a has a broad, asymmetric fluorescence emission peak centered at 341 nm. Binding of Ca2+ of Mg2+ to DNase shifted the peak to 342 nm and caused a 10% fluorescence enhancement. Half of the maximum change occurred at 6 x 10(-5) M Ca2+ or at 6 x 10(-4) M Mg2+. The change in fluorescence is most likely due to a conformational change in DNase which occurs when Ca2+ or Mg2+ is bound to the nonspecific tight Ca2+ binding site on DNase. The kinetics of the fluorescence change, followed by stopped flow techniques, show a fast phase (65%) and a slow phase (35%). At 1 mM Ca2+, the half-time for the fast phase is 17 ms, and for the slow phase, 3.5 min. Both phases of the reaction are first order in DNase and independent of Ca2+ at concentrations above 1 mM. DNase incubated with Ca2+ undergoes a slow (t 1/2 approximately equal to 6 min) 1.5-fold increase in activity. This activation follows pseudo-first order kinetics and is not due to the presence of additional Ca2+ in the substrate. The simplest hypothesis which accounts for these data, and previously reported studies on the effect of Ca2+ on DNase, is that DNase exists in 3 conformational states at 25 degrees C and pH 7.5. The kinetics are consistent with a mechanism which can be diagrammed as: (formula: see text) where A, A, and B represent three conformational states of DNase.
D002118:Calcium / Q000494:pharmacology*; D003851:Deoxyribonucleases / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008433:Mathematics; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D003851:Deoxyribonucleases; C019328:deoxyribonuclease A; D008274:Magnesium; D002118:Calcium