Conformational similarities between one-chain and two-chain tissue plasminogen activator (t-PA): implications to the activation mechanism on one-chain t-PA.
Tissue plasminogen activator (t-PA) is an exceptional serine protease, because unlike most other serine protease zymogens single-chain tissue plasminogen activator (sct-PA) possesses a substantial amount of proteolytic activity. The unusual reaction of sct-PA afforded the opportunity to directly compare the active site environment of sct-PA and two-chain tissue plasminogen activator (tct-PA) in solution through the application of a series of nitroxide spin labels and fluorophores. These labels, which have been previously shown to covalently label the catalytic serine of other serine proteases, inactivated both sct-PA and tct-PA. The labels can be divided into two classes: those which form tetrahedral complexes (sulfonates) and those which form trigonal complexes (anthranilates). Those which formed tetrahedral complexes were found to be insensitive to structural differences between sct-PA and tct-PA at the active site. In contrast, those which formed trigonal complexes could differentiate and monitor the sct-PA to tct-PA conversion by fluorescence spectroscopy. Models of the structure of sct-PA and tct-PA were constructed on the basis of the known X-ray structures of other serine protease zymogen and active enzyme forms. One of the nitroxide spin labels was modeled into the sct-PA and tct-PA structures in two possible orientations, both of which could be sensitive to structural differences between sct-PA and tct-PA. These models formed the structural rationale used to explain the results obtained with the "tetrahedral" and "trigonal" probes, as well as to offer a possible explanation for the unique reactivity of sct-PA.
Nienaber
V L
VL
Department of Chemistry, Ohio State University, Columbus 43210.
Young
S L
SL
Birktoft
J J
JJ
Higgins
D L
DL
Berliner
L J
LJ
D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D013113:Spin Labels; D010959:Tissue Plasminogen Activator