THERMODYNAMICS AND KINETICS OF SINGLE RESIDUE REPLACEMENTS IN 17 OVOMUCOID THIRD DOMAINS: EFFECT ON INHIBITOR INTERACTIONS WITH 3 SERINE PROTEINASES
Abstract
Avian ovomucoid third domains are Kazal family protein inhibitors of serine proteinases. From a list of over 30 amino acid sequences of these domains, 15 were selected which contained only one or a few amino acid substitutions compared to another sequence. The thermodynamic and kinetic constants were determined for these inhibitors, each interacting with (alpha)-chymotrypsin, elastase I and subtilisin Carlsberg. The compared constants for singly substituted pairs provides the binding energy difference only for the one side chain substitution. The results show: (1) All residue substitutions in positions of potential contact in the enzyme-inhibitor complex can change the binding reaction. (2) Changes in non-contact, surface residues are without effect. The results were rationalized on the basis of computer confrontation, 3-dimensional x-ray structure models of the complexes. Binding constant changes were largest when hydrophobic inhibitor side chains were substituted by charged or hydrophilic groups; substitutions by other hydrophobic side chains produced lesser effects. Binding constants were often differentially affected with respect to the three enzymes. The limited data set which was generated provides the basis for a predictive sequence to reactivity algorithm. With the knowledge of the binding constants for a number of inhibitors containing single amino acid substitutions, a prediction of a binding constant can be made for an untested inhibitor from only its amino acid sequence. Already explanations can be made why several sequences result in poor inhibitors. Several characteristics of Kazal inhibitors suggest that they are suitable for the basis of such an algorithm. Analysis of amino acid variability for ovomucoid sequences shows that the surface, contact residues are most variable. Internal, structurally important residues are strongly conserved. Thus, with further sequencing, more natural variants will become available. The internal inhibitor structure will provide a rigid framework on which to test the substitutions.
Degree
Ph.D.
Subject Area
Biochemistry
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