Volume 272, Number 1, Issue of January 3, 1997 pp. 28-31
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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COMMUNICATION:
Converting Tissue Type Plasminogen Activator into a Zymogen
IMPORTANT ROLE OF Lys¹⁵⁶

(Received for publication, October 28, 1996, and in revised form, November 6, 1996)

From The Scripps Research Institute, Department of Vascular Biology, La Jolla, California 92037

In stark contrast to most other members of the chymotrypsin family of serine proteases, tissue type plasminogen activator (t-PA) is not synthesized and secreted as a true zymogen. Instead, single-chain t-PA exhibits very significant catalytic activity. Consequently, the zymogenicity, or ratio of the catalytic efficiencies of the mature, two-chain enzyme and the single-chain precursor, is only 3-9 for t-PA. Both we and others have previously proposed that Lys¹⁵⁶ may contribute directly to this exceptional property of t-PA by forming interactions that selectively stabilize the active conformation of the single-chain enzyme. To test this hypothesis we created variants of t-PA in which Lys¹⁵⁶ was replaced by a tyrosine residue. As predicted, the K156Y mutation selectively suppressed the activity of the single-chain enzyme and thereby substantially enhanced the enzyme's zymogenicity. In addition, however, this mutation produced a very dramatic increase in the ability of single-chain t-PA to discriminate among distinct fibrin co-factors. Compared with wild type t-PA, one of the variants characterized in this study, t-PA/R15E,K156Y, possessed substantially enhanced response to and selectivity among fibrin co-factors, resistance to inhibition by plasminogen activator inhibitor type 1, and significantly increased zymogenicity. The combination of these properties, and the maintenance of full activity in the presence of fibrin, suggest that the R15E,K156Y mutations may extend the therapeutic range of t-PA.

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