Reaction of proteinases with .alpha.2-macroglobulin: rapid-kinetic evidence for a conformational rearrangement of the initial .alpha.2-macroglobulin-trypsin complex

Abstract

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

Bait Region Involvement in the Dimer-Dimer Interface of Human α2-Macroglobulin and in Mediating Gross Conformational Change

“…Even when conformational change is brought about by proteinase cleavage of the bait region, there is evidence from the greatly lowered efficiency of proteinase trapping (Table I) that the structural rearrangements may have been slowed down. Such changes in efficiency of proteinase trapping as a function of the relative rates of cleavage and conformational change have been documented for plasma ␣ 2 M and trypsin (27,28). This suggests that when the thiol esters alone are cleaved, the conformational change that occurs cooperatively between noncovalently interacting subunits across the dimer-dimer interface involves a reorganization of the bait region-bait region contact area.…”

The kinetics of conformational changes of α2‐macroglobulin determined by time resolved X‐ray solution scattering

“…This proved that the rate of conformational change was not limited by the encounter of proteinase but by one of the first-order reactions following the encounter, including the conformational change itself. This rate of the limiting step may correspond to k, proposed by Strickland et al [13]. This means that the slower half of the TNS (6-(p-toluidino)-2-naphthalenesulfonic acid) fluorescence increase had corresponded to the gross conformational change.…”

Studies on the Refolding of Egg White Lysozyme Denatured by Urea Using "Phase Diagram" Method of Fluorescence

“…According to reference, 32 lysozyme activity was analyzed at 25 ℃ by following the decrease in the absorbance of a reaction solution at 450 nm. The reaction process was carried out by adding 0.1 mL of enzyme solution into 1 mL of 0.25 mg/mL micrococcus lysodeikticus suspension in 0.06 mol/L potassium phosphate (pH 6.2).…”