Role of the zinc atom in the activity and conformational stability of serratial 56K protease. A fluorometric study.
We found a zinc content of 1 atom per mol in 56K protease (produced by the gram-negative bacterium Serratia marcescens kums 3958) by the neutron activation method. Selective removal of the functional zinc ion from 56K protease with 10 mM tetraethylenepentamine (TEP) in the presence of 10 mM CaCl2 at room temperature at pH 8.0 resulted in complete loss of the enzyme activity without affecting the lambda max of the enzyme, but with a 10% decrease in fluorescence intensity. Ethylenediaminetetraacetic acid (EDTA) at 10 mM gave similar results at pH 5.0, but produced a 30% decrease in fluorescence intensity. Melting profile experiments carried out by monitoring the fluorescence intensity at 333 nm showed a melting temperature (Tm) of about 61.0, 62.5, and 60.0 degrees C at pH 5.0, 7.0, and 8.0, respectively. Tm became much lower upon removal of the zinc ion, falling to 42.5 degrees C with 10 mM TEP or to 47.5 degrees C with 10 mM EDTA in the presence of 10 mM CaCl2. The CD data showed very little change in conformation upon removal of the zinc ion under physiological conditions, but the conformation appears to be readily disrupted to a denatured form by changing either temperature or pH or by exposure to denaturants. These results suggest that a single zinc ion is essential for the enzyme function and contributes to the conformational integrity of the enzyme, but tryptophan residues appear to be not directly related to the enzyme activity.
D002942:Circular Dichroism; D010450:Endopeptidases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008666:Metalloendopeptidases; D009501:Neutron Activation Analysis; D011487:Protein Conformation; D011489:Protein Denaturation; D012706:Serratia marcescens / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D015032:Zinc / Q000032:analysis*
D010450:Endopeptidases; D008666:Metalloendopeptidases; C044194:microbial metalloproteinases; D015032:Zinc