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, 77636-7643]. In this model, once an initial 1:1 alpha-2M-proteinase complex forms, the complex either can react with a second proteinase molecule or can undergo a conformational rearrangement that generates a complex greatly reduced in its ability to bind additional proteinase. The detection of two kinetic phases in the present study and the excellent agreement between the range of 1-2 s-1 predicted from modeling experiments for the magnitude of this conformational change and the value of 1.4 s-1 obtained for the slow phase provide evidence supporting this model.