The specificity of ovomucoid third domain at residues other than P1
Abstract
The main focus of this work is the interaction of turkey ovomucoid third domain (OMTKY3) and its many variants, a standard mechanism canonical inhibitor, with six serine proteinases of primarily hydrophobic specificity. We are interested in determining the role of each contact residue in enzyme-inhibitor association. Complete sets of all twenty amino acid variants at each contact position (based on a consensus of three crystal structures) have been expressed and the free energies of association (ΔG°a) measured at pH 8.3. The role of the P4 and P1′ residues are discussed in detail while that of other contact residues is briefly reviewed. In addition to specificity, the contribution of each position to overall enzyme inhibitor association can be calculated and compared. Since all standard mechanism inhibitors are expected to interact in the same manner to a given enzyme, energetic changes observed in the OMTKY3 frame should prove to be additive for other inhibitors. Insertion of a residue into a hydrophobic pocket leads to a large shift in the ionization constant of the residue which can be described by two pKs: that in free inhibitor and in complex. The pK of ionizable P1 residues in OMTKY3 were determined by NMR. Excellent agreement was found between different inhibitors as well as with substrates. Once the pK in complex has been determined, binding of an ionizable residue can now be considered as either fully protonated or unprotonated. Comparisons of nonionizable residues shows that the difference in ΔG°a is pH independent allowing us to extrapolate ΔG° a binding data at pH 8.3 to other pHs. Additivity also allows us to predict the reactivity of an inhibitor from its sequence alone. This has significant implications for the increasing role of genomics where the sequence but not the protein is available. The ΔG° a of any Kazal inhibitor family sequence can be regarded as the sum of free energy changes from each contact position that differs from OMTKY3. ΔG° a data of natural ovomucoids affords us a test set for our predictions. This approach can be extended to other inhibitor families once an appropriate baseline has been established.
Degree
Ph.D.
Advisors
Laskowski, Purdue University.
Subject Area
Biophysics|Biochemistry
Off-Campus Purdue Users:
To access this dissertation, please log in to our
proxy server.