ACTIVE PROTEOLYTIC DERIVATIVE OF THE ALPHA-SUBUNIT OF TRYPTOPHAN SYNTHASE - IDENTIFICATION OF THE SITE OF CLEAVAGE AND CHARACTERIZATION OF THE FRAGMENTS

Previous studies showed that the limited tryptic proteolysis of theα 2β2 complex of tryptophan synthase yields an active α2β2 derivative which can be resolved into an active β2 dimer and an active a derivative termed α' [Miles, E. W., & Higgins, W. (1978) J. Biol. Chem. 253, 6266-6269], α' has now been resolved into two fragments,α-1 and α-2, by gel filtration in 6 M urea. A site of cleavage of the α chain at arginine-188 has been identified by amino acid analysis and N-terminal sequence analysis of the two fragments; α-1 is the amino-terminal part of the α chain and α-2 is the carboxy-terminal fragment. Circular dichroism spectra in the far-UV show that the separate α-1 and α-2 fragments have ordered structure after removal of urea. Further evidence that the α-1 fragment has an ordered, folded structure results from solvent perturbation and circular dichroism studies of tyrosyl residues. Whereas neither of the fragments alone has catalytic activity or the ability to bind the substrate analogue indolepropanol phosphate or the holo β2 dimer, mixing of the two fragments restores those binding properties and catalytic activity. We conclude that the two fragments are separate folding domains which must interact to form functional enzyme. In addition, we conclude that arginine-188 must be readily accessible to trypsin in the α2β2 complex, since cleavage at this bond is the predominant result of limited tryptic proteolysis of the α2β2 complex. © 1979, American Chemical Society. All rights reserved.