Energetics of the interactions of ovumucoid third domain variants with different serine proteinases

Wuyuan Lu, Purdue University

Abstract

Turkey ovomucoid third domain (OMTKY3) is a powerful inhibitor of most serine proteinases that prefer a neutral residue at P$\sb1$ (Schechter & Berger, 1967), including bovine $\alpha$-chymotrypsin (CHYM), porcine pancreatic elastase (PPE), human leukocyte elastase (HLE), subtilisin Carlsberg (SUBT), Streptomyces griseus proteinase A and B (SGPA and SGPB). Based on three X-ray crystal structures of OMTKY3 in complexes with CHYM, HLE and SGPB (Read et al., 1983; Bode et al., 1986; Fujinaga et al., 1987), a consensus contact set comprising 12 residues was defined (Apostol et al., 1993), among which the P$\sb1$ residue, with its side chain buried in the S$\sb1$ pocket of the enzyme, is the primary inhibitory specificity determinant. The fundamental task in studying proteinase-protein inhibitor interactions is to establish the first order sequence to reactivity algorithm, so that equilibrium association constant, K$\sb{\rm a}$, can be predicted for an inhibitor based on its amino acid sequences alone. This will, in turn, enable us to design new inhibitors to target enzymes of interest. In the process of doing this, we expressed wild-type and 38 alternative variants of OMTKY3 at P$\sb1$ and P$\sb3\sp\prime$, and measured K$\sb{a}$ values of these variants with six enzymes. An extremely broad dynamic range of K$\sb{\rm a}$, spanning 6-8 orders of magnitude, was observed at P$\sb1$. With the aid of 4 synthetic P$\sb1$ variants whose straight aliphatic side chains differ only in length (Bigler et al., 1993), many comparisons within the P$\sb1$ set were made regarding the energetic consequences of introducing branching, heteroatoms and charges into the S$\sb1$ pocket (Lu et al., 1994). To demonstrate the generality of our conclusions, the results also were compared with the data in the literature, and a strong positive correlation was found. Extensive crystallographic studies of the whole set of P$\sb1$ variants in complexes with several enzymes are underway, to elucidate the structural basis, which will eventually provide us with a deep insight into the energetics of amino acid recognition and protein-protein interactions.

Degree

Ph.D.

Advisors

Laskowski, Purdue University.

Subject Area

Biochemistry

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