To determine whether formation of the stable complex between a
serpin and a target
proteinase involves a major
translocation of the
proteinase from its initial position in the noncovalent Michaelis complex, we have used
fluorescence resonance energy transfer to measure the separation between
fluorescein attached to a single
cysteine on the
serpin and tetramethylrhodamine conjugated to the
proteinase. The interfluorophore separation was determined for the noncovalent Michaelis-like complex formed between alpha 1-proteinase inhibitor (Pittsburgh variant) and anhydrotrypsin and for the stable complex between the same
serpin and
trypsin. A difference in separation between the two
fluorophores of approximately 21 A was found for the two types of complex. This demonstrates a major movement of the
proteinase in going from the initial noncovalent encounter complex to the kinetically stable complex. The change in interfluorophore separation is most readily understood in terms of movement of the
proteinase from the
reactive center end of the
serpin toward the
distal end, as the covalently attached
reactive center loop inserts into
beta-sheet A of the
serpin.
