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coagulation, fibrinolysis, the complement reaction, and others. The key point here is that a signal can be specifically and irreversibly amplified every time a downstream inactive enzyme precursor is activated. Recent work, to be presented in the paper to follow (8), has demonstrated a specific role of the pro segment of activation, which early on was regarded a throwaway piece but in certain cases can act as an intramolecular inhibitor and as an intramolecular chaperon that assures proper folding of the active enzyme (10).

Certain generalizations have emerged from these and related investigations. If I may borrow a page from Nancy Thornberry (11), most proteases are synthesized as inactive precursors (zymogens) that require limited proteolysis for activation. Because proteolysis is irreversible under physiological conditions, the generation of the uncleaved precursor requires de novo synthesis. All active proteases, including those that activate zymogens, are regulated by specific inhibitors. However, some protease precursors can regulate their own activation, e.g., trypsinogen, whereas others, e.g., plasminogen, do not require peptide bond cleavage for their activation. Proteolytic processing, like all proteolytic reactions, requires unique combinations of primary, secondary, and tertiary structures to permit interaction with substrate so as to form the reactive enzyme-substrate intermediate.

Let me now make a major leap in time and discuss in brief how we reached the current era of research on proteolytic enzymes and what we can expect in the millennium that we are about to enter. Two major factors have expanded our conceptual horizons and endowed us with experimental tools of previously unimaginable powers of resolution. One factor is the application of the newly emerging concepts and methodologies of molecular and cell biology, such as DNA cloning and sequencing, site-directed mutagenesis, gene amplification, gene knockouts, phage display, and the wealth of information yielded by genomics research generally. The other major impetus came from a group of newly developed concepts and experimental approaches to the structure and function of proteins by mass spectroscopy (12), multidimensional NMR, and the use of computers for the prediction of protein structure based on various types of algorithms. To these one might add the methods of combinatorial chemistry as applied to proteins to scan and identify protein ligands of physiological significance. Although we are still far from understanding the rules of the in vivo folding of nascent polypeptide chains, the challenge lies in deriving the function of a protein from its known chemical and biological parameters and in learning how to design proteins of predetermined physiological properties. All of these developments, singly and in combination, expand our horizons and the goals that we are setting for their application to biology and medicine.

The importance of proteolytic enzymes to the understanding of vital biological tasks is perhaps best illustrated by current trends in the study of viral proteases (13). In every known instance, the timing, placement, and mode of action of the virus encoded protease are somehow adapted to the conditions under which it operates within the viral environment. Two examples follow: in herpes viruses such as cytomegalovirus, the structure of the protease reveals a catalytic triad of His/His/Ser instead of the conventional Asp/His/Ser of the mammalian serine proteases and a single beta barrel structure per monomer instead of two in the mammalian serine proteases (13). Analogously, in adeno viruses the cysteine protease contains a Glu/His/Cys catalytic triad characteristic of cysteine proteases, but the seven alpha helices and a single five-stranded beta sheet are not seen in the parent protease (papain). In either case, the examples given demonstrate the ability of the virus proteases to adapt themselves to the evolution of functions within the limits of compatible protein structures (13).

Other rapidly expanding areas of biological research involving well known proteases include those of apoptosis, the mediation of thrombin signaling by protease activated receptors, proteolytic processing in cholesterol metabolism, in the cell cycle, and the many others included in this issue of the Proceedings.

It is no coincidence that industry and academia are almost equally represented in this audience, because intense cooperation between both is essential if we are to reap the full benefits of the advances and discoveries in both basic and applied research.

1. Northrop, J.H., Kunitz, M. & Herriott, R.M. (1938) Crystalline Enzymes (Columbia Univ. Press, New York).

2. Neurath, H. (1995) Protein Sci. 4, 1939–1943.

3. Barrett, A.J., Rawlings, N.D. & Woessner, J.F. (1998) in Handbook of Proteolytic Enzymes (Academic, New York), pp. xiii–xxix.

4. Kitamoto, Y., Yuan, X., Wu, Q., McCourt, D.W. & Sadler, J.E. (1994) Proc. Natl. Acad. Sci. USA 91, 7588–7592.

5. Perona, J.J. & Craik, C.S. (1995) Protein Sci. 4, 337–360.

6. Higaki, J.N., Fletterick, R.J. & Craik, C.S. (1992) Trends Biochem. Sci. 17, 100–104.

7. Klemba, M., Gardner, K.H., Marino, S., Clarke, N.D. & Regan, L. (1995) Struct. Biol. 2, 368–373.

8. Salvesen, G.S. & Dixit, V.M. (1999) Proc. Natl. Acad. Sci. USA 96, 10964–10967.

9. Groll, M., Heinemeyer, W., Jäger, S., Ullrich, T., Bochtler, M., Wolf, D.H. & Huber, R. (1999) Proc. Natl. Acad. Sci. USA 96, 10976–10983.

10. Cunningham, E.L., Jaswal, S.S. & Agard, D.A. (1999) Proc. Natl. Acad. Sci. USA 96, 11008–11014.

11. Thornberry, N.A. & Lazebnik, Y. (1998) Science 281, 1312–1316.

12. Cohen, S.L. (1996) Structure (London) 4, 1013–1016.

13. Babé, L.M. & Craik, C.S. (1997) Cell 91, 427–430.

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