In support of the trap hypothesis. Chymotrypsin is not rigidly held in its complex with human alpha 2-macroglobulin.
Complexes (2:1) of chymotrypsin with human alpha 2-macroglobulin have been prepared in the presence of 200 mM methylamine such that 90% of the chymotrypsin remains noncovalently bound to the alpha 2-macroglobulin. Reaction of this complex with the active-site-directed spin-labeling reagent 4-[(ethoxyfluorophosphinyl)oxy]-2,2,6,6-tetramethylpiperidinyl+ ++-1-oxy results in nitroxide labeling of the active-site serine residue of the complexed chymotrypsin. Electron spin resonance (ESR) spectra of this complex were recorded at 275 K in buffer and at 263 K in 50% glycerol. At 263 K in 50% glycerol the spectrum is that expected for a rigid glass, whereas at room temperature the ESR spectrum shows that the chymotrypsin is only slightly immobilized compared with free spin-labeled chymotrypsin. These findings are discussed in relation to possible models of inhibition of protease activity by alpha 2-macroglobulin. It is concluded that the trap mechanism of Barrett and Starkey [Barrett, A. J., & Starkey, P. M. (1973) Biochem. J. 133, 709-724] is the only model currently considered that can account for the present findings.
D002918:Chymotrypsin / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008744:Methylamines / Q000494:pharmacology; D008962:Models, Theoretical; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D000511:alpha-Macroglobulins / Q000302:isolation & purification / Q000378:metabolism*
D008744:Methylamines; D000511:alpha-Macroglobulins; C027451:methylamine; D002918:Chymotrypsin