The effect of the one-chain to two-chain conversion in tissue plasminogen activator: characterization of mutations at position 275.
Tissue plasminogen activator (t-PA) is homologous to other serine proteases and contains an apparent activation cleavage site at arginine 275. It has been demonstrated that this arginine-275 can be replaced with either glutamic acid (Tate, K. M., Higgins, D. L., Holmes, W. E., Winkler, M. E., Heyneker, H. L., and Vehar, G. A. Biochemistry 26, 338-343, 1987) or glycine (Peterson, L. C., Johannessen, M., Foster, D., Kumar, A., and Mulvihill, E. Biochim. Biophys. Acta 952, 245-254, 1988; Boose, J. A., Kuismanen, E., Gerard, R., Sambrook, J. and Gething, M.-J. Biochemistry 28, 635-643, 1989) so that the product of the plasminogen activation reaction, plasmin, can no longer hydrolyze the one-chain form of t-PA to the two-chain form. These "one-chain" t-PA variants had diminished activity, compared to wild-type t-PA, in the absence of a cofactor, but in the presence of the fibrin(ogen) cofactor the two variants had activity similar to wild-type t-PA. In order to compare the effects of all possible substitutions, t-PA variants with each of the other nineteen amino acids besides arginine at position 275 were produced by site-directed mutagenesis. All were recovered from cell culture supernatants completely in the one-chain form, except for R275 (wild-type) and R275K, which were partially converted to the two-chain form. These latter two species could be completely converted to the two-chain form by plasmin. In addition, these two forms showed significantly more plasminogen activating activity in the absence of a fibrin(ogen) cofactor, compared to the other 18 variants. In the presence of a cofactor, all of the t-PA mutants had plasminogen activating activity equivalent to wild-type t-PA, except for R275C. The R275C t-PA had comparatively less clot lysis and fibrin binding activity as well. Presumably the new cysteine in this variant was involved in a mixed disulfide or caused misfolding of the molecule resulting in decreased activity. The difference in the plasminogen activating activity of one- and two-chain forms of t-PA was investigated by determining the apparent Michaelis constants and the apparent turnover numbers for R275E t-PA, which remains in the one-chain form throughout the assay, and two-chain R275 t-PA. The kinetic constants were measured in both the presence and the absence of plasmin-digested fibrinogen.(ABSTRACT TRUNCATED AT 400 WORDS)
Higgins
D L
DL
Department of Cardiovascular Research, Genentech, Inc., South San Francisco, CA 94080.
Lamb
M C
MC
Young
S L
SL
Powers
D B
DB
Anderson
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D004789:Enzyme Activation; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D010958:Plasminogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin