States of tryptophyl residues and stability of recombinant human matrix metalloproteinase 7 (matrilysin) as examined by fluorescence.
States of tryptophyl residues and stability of human matrilysin were studied. The activation energy for the thermal inactivation of matrilysin was determined to be 237 kJ/mol, and 50% of the activity was lost upon incubation at 69 degrees C for 10 min. The activity was increased by adding NaCl, and was doubled with 3 M NaCl. Denaturation of matrilysin by guanidine hydrochloride (GdnHCl) and urea was monitored by fluorescence change of tryptophyl residues. Half of the change was observed at 2.2-2.7 M GdnHCl, whereas no change was observed even with 8 M urea. Half of the inactivation was induced at 0.8 M GndHCl and at 2 M urea. The presence of an inactive intermediate with the same fluorescence spectrum as the native enzyme was suggested in the denaturation. Matrilysin contains four tryptophyls, and their states were examined by fluorescence-quenching with iodide and cesium ions and acrylamide. No tryptophyls in the native enzyme were accessible to I(-) and Cs(+), and 2.4 residues were accessible to acrylamide. Based on the crystallographic study, Trp154 is water-accessible, but it should be in a crevice not to contact with I(-) and Cs(+). All tryptophyls in the GdnHCl-denatured enzyme were exposed to the quenchers, while a considerable part was inaccessible in the urea-denatured one.
Inouye
K
K
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. inouye@kais.kyoto-u.ac.jp
Tanaka
H
H
Oneda
H
H
D017364:Collagenases / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D019791:Guanidine / Q000378:metabolism; D006801:Humans; D020783:Matrix Metalloproteinase 7 / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012965:Sodium Chloride; D014364:Tryptophan; D017364:Collagenases; D020783:Matrix Metalloproteinase 7; D019791:Guanidine