Education
1969        B.S.        State University of New York at Stony Brook
1975        Ph.D.      Duke University, Durham, NC

Research Area of Interests
Structure and function of Serine Proteases and their inhibitors

Summary of Current Research Aims
The major research interests of the laboratory are in the structure, function, and regulation of serine proteases and serine protease inhibitors. Research concentrates on two human serine proteinases termed tryptase and chymase. Both are found in high abundance within the secretory granules of inflammatory cells known as mast cells. High yield recombinant expression of both proteases has made it possible to study their structural and functional properties in detail. The x-ray crystal structure of human chymase bound to a peptide inhibitor was determined; this structure allows for an understanding of the enzyme at the molecular level.

Both proteases have a number of unusual properties that make their study of interest. Tryptase is a tetramer composed of 4 catalytic subunits, has no physiological inhibitor to regulate its proteolytic activity, and spontaneously inactivates in solutions of physiological pH, salt and temperature, unless in the presence of a highly sulfated GAG such as heparin. The structural reasons for tryptase's functional instability and how sulfated GAGs stabilize the protease are being pursued. Rescue of inactivated tryptase by heparin, small strong-binding inhibitors, and high salt suggests that the inactivation process is reversible and is produced by a limited structural change.

The biological role of chymase (and tryptase) is uncertain. The x-ray crystal structure of the chymase-peptide inhibitor complex demonstrates an extraordinary number of charged residues in the active site as well as the unusual orientation of a highly conserved phenylalanine residue. These residues may be factors in determining the substrate specificity of the protease. Their role in the hydrolysis of substrates and in the interaction of chymase with protease inhibitors is being explored using site directed-mutagenesis.

The mechanism of protease inhibition by members of the serpin family is controversial appearing to depend on the trapping of the enzyme in a covalent complex resulting from incomplete hydrolysis of the reactive center peptide bond. Cleavage of the inhibitor proceeds only to the formation of the serpin acyl-enzyme complex, a species believed comparable to the acyl-enzyme intermediate in substrate hydrolysis. Inactivation of chymase by certain serpins deviates from this mechanism in two ways: 1) inactivation occurs with an unusually high stoichiometry of inhibition indicating a partitioning step that produces cleaved-inactivated inhibitor in a substrate-like manner, and 2) the rate constant of inhibition does not reveal the kind of pH dependence expected if formation of the serpin acyl-enzyme complex is rate limiting. Chymase-serpin complexes are extremely stable and physical studies show that the enzyme is altered structurally in the complex. Establishing the critical step producing the partitioning of the reaction, the reason for the unexpected pH dependence, and the specific structural changes in chymase producing stable complex formation are being explored to define the mechanism of serpin inhibition.

Dr. Schechter's Lab Personnel:
Trevor Selwood, Ph.D., Senior Research Scientist
Eun-Jung Choi, Research Specialist


Selected Publications