Reaction of proteinases with alpha 2-macroglobulin: rapid-kinetic evidence for a conformational rearrangement of the initial alpha 2-macroglobulin-trypsin complex.
The kinetics of the reaction of trypsin with alpha 2M were examined under pseudo-first-order conditions with excess inhibitor. Initial studies indicated that the fluorescent dye TNS is a suitable probe for monitoring the reaction over a wide concentration range of reactants. Titration experiments showed that the conformational changes associated with the binding of trypsin to alpha 2M result in an increased affinity of the inhibitor for TNS. Two distinct phases were observed when this dye was used to monitor the progress of the reaction. Approximately half of the fluorescence signal was generated during a rapid phase, with the remainder generated during a second, slower phase. The observed pseudo-first-order rate constant of the first phase varied linearly with the concentration of alpha 2M up to the highest concentration of inhibitor used, whereas the rate constant of the second phase was independent of alpha 2M concentration. The data fit a mechanism in which the association of trypsin with alpha 2M occurs in two consecutive, essentially irreversible steps, both leading to alterations in TNS fluorescence. The initial association occurs with a second-order rate constant of (1.0 +/- 0.1) X 10(7) M-1 s-1 and is followed by a slower, intramolecular conformational rearrangement of the initial complex with a rate constant of 1.4 +/- 0.2 s-1. The data are consistent with a previously proposed model for the reaction of proteinases with alpha 2M [Larsson et al. (1989) Biochemistry 28, 7636-7643].2+ this model, once an initial 1:1 alpha 2M-proteinase
Strickland
D K
DK
Department of Biochemistry, Holland Laboratory, American Red Cross, Rockville, Maryland 20855.
Larsson
L J
LJ
Neuenschwander
D E
DE
Björk
I
I
D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D000511:alpha-Macroglobulins; D010450:Endopeptidases; D014357:Trypsin