Structure-function investigations of proteinase inhibitors and proteinases

The specific aims of this work have been to determine important structural and dynamic features of turkey ovomucoid third domain (OMTKY3), a well-studied, canonical, standard-mechanism, protein inhibitor of proteinases, and to investigate its mechanism of interaction with chymotrypsin. Chapter 2 describes NMR investigations of 32 variants of OMTKY3 with Asp, Glu, His, or Lys at one of eight of the nine contiguous contact positions of the inhibitor. Results include the measurement of 31 of the 32 pK_a values for these residues and the detection of an intramolecular hydrogen bond and an electrostatic interaction within the P₂, P₁′ P₃′ triad of the inhibitor. Chapter 3 examines the physical basis for the unusually small hydrolysis constants observed for OMTKY3 and related proteins. NMR relaxation investigations were used to probe the role of internal dynamics in this equilibrium, and measurements of chemical constants and spin-spin couplings across hydrogen bonds were used to evaluate structural contributions. The results ruled out an entropic contribution and assigned the change to an enthalpic contribution. Chapter 4 presents a detailed analysis of a conformational transition in OMTKY3. The three-dimensional structures of the two interconverting states were determined by NMR spectroscopy under conditions where they coexist, and the kinetics and thermodynamics of this process were examined. The results show that the two states differ by cis/trans isomerization of a prolyl peptide bond, which is coupled to rearrangements of the conformations of two disulfide bridges. Chapter 5 describes an investigation of changes in OMTKY3 that accompany the formation of a complex with bovine chymotrypsin A. The results demonstrate that the peptide bond remains intact in the complex and that its hybridization is trigonal with a possible slight progression toward tetrahedral. The final chapter (Chapter 6) describes the successful production and stable isotope labeling of a vertebrate serine proteinase for NMR investigations. Human chymotrypsinogen B1 (hCtgB) was subcloned and expressed in Pichia pastoris. The results indicate that it will be possible to use multinuclear NMR spectroscopy to investigate active site residues in this protein system.