Conformational change at the active center of serine-proteases upon their binding to alpha 2-macroglobulin.
The inhibition rates and spectral characteristics of 2 probes specific for the active-site serine residue of proteases were examined for evidence of conformational change of the proteases upon their binding to alpha 2-macroglobulin (alpha 2M). Elastase, chymotrypsin, trypsin, and plasmin were reacted with (7-nitrobenz-2-oxa-1,3-diazole) aminoethyl- and aminopentyl methylphosphonofluoridate. The inhibition rate constants depend on the chain length of the aminoalkyl moiety of the probe and range from 10(5) to 10(4) M-1 min-1 for elastase and chymotrypsin. They are significantly modified when the proteases are stoichiometrically bound to alpha 2M. The absorption maximum of the chromophore appears in the range of 460-470 nm and 475-480 nm for the aminoethyl- and aminopentyl- conjugates, respectively. The fluorescence emission is maximal around 530 nm with a low quantum yield of about 3%. These spectral characteristics are altered in different ways by the covalent or non-covalent binding mode of the protease to alpha 2M. Finally, the CD spectrum of the NBD aminoethyl and aminopentyl elastase and chymotrypsin conjugates exhibits intense optical activity in the absorbing band of the NBD-moiety. These chiral properties are greatly altered upon binding of the protease to alpha 2M. All these results strongly suggest a conformational change in the protease at its active center upon its binding to alpha 2M; this conformational change could be taken into account to explain the alteration of the catalytic properties of the alpha 2M-bound proteases.
Martini
J L
JL
U. 219 INSERM, Institut Curie-Biologie, Orsay, France.
Pochon
F
F
D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D005341:Fibrinolysin / Q000378:metabolism; D009904:Optical Rotation; D010196:Pancreatic Elastase / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012524:Sarin / Q000031:analogs & derivatives / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism; D000511:alpha-Macroglobulins / Q000378:metabolism*
D015842:Serine Proteinase Inhibitors; D000511:alpha-Macroglobulins; C044737:5-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminoethylmethylphosphonofluoridate; C060280:(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminopentylmethylphosphonofluoridate; D012524:Sarin; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D014357:Trypsin; D005341:Fibrinolysin