The energetic effects of the P2 residue in standard mechanism inhibitors on the strength of association with different serine proteinases

Michael Rabia Ranjbar, Purdue University

Abstract

The Turkey Ovomucoid Third Domain Inhibitor (OMTKY3) is believed to represent an ideal model for studying the interactions of all standard mechanism inhibitors (STM) with serine proteinases. The effects of single mutations at the P 1 position of OMTKY3 were previously characterized and found to predict mutations in other STM's and substrates at the same position. (The P 1 position is one of twelve contact residues defined in OMTKY3 numbering outward from the reactive site bond cleaved P1-P1 ′). Given that there are 20 different families of STM's and that many of these inhibitors are of physiological importance, it is of interest to know whether mutations in other contact positions in OMTKY3 can also predict mutations among STM's. In this thesis we measured the Ka of 18 P2 variants of OMTKY3 with six different serine proteinases at pH 8.3 and five of the six serine proteinases at pH 5.0. In addition, we determined the pH dependencies of several P1 and P2 variants of OMTKY3 for the same serine proteinases. We found that mutations at the P2 position (other than wild type Thr) caused a general lowering in the Ka values of all mutants (at pH 8.3) for all enzymes studied except Carlsberg subtilisin (CARL). Previously it was believed that mutations other than P2 Thr and Ser in OMTKY3 could not predict the same mutations in other STM's. However, by comparing the P2 variant Ka values with chymotrypsin (CHYM) at pH 8.3 with a cyclic Bowman Birk peptide standard mechanism inhibitor (cBBI), we were able to determine that the P2 mutations for hydrogen bonding residues in OMTKY3 could predict the energetic effects of the same mutations in cBBI. In addition, we found that the P2 mutations in OMTKY3 could not predict the energetic effects of the same mutations in substrates. We developed a method to determine the pK of ionizable residues in complex at the P 1 position in OMTKY3. This method was reapplied to the P2 position to confirm that the P2 residue does interact with the His57 in most of the serine proteinases we study and assign an energetic effect to that interaction in CHYM.

Degree

Ph.D.

Advisors

Laskowski, Purdue University.

Subject Area

Biochemistry|Physical chemistry|Molecular biology|Chemistry

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