EFFECT OF AMINO ACID REPLACEMENTS IN OVOMUCOID THIRD DOMAINS UPON THEIR ASSOCIATION WITH SERINE PROTEINASES (EQUILIBRIUM CONSTANT, ADDITIVITY)

SOON JAE PARK, Purdue University

Abstract

The general objective of this work was to contribute to the formulation of an algorithm which will allow us to predict the enzyme-proteinase inhibitor interaction from the amino acid sequence of the inhibitor. To this end, the equilibrium constants, K(,a)('obs), for the interaction of 67 different ovomucoid third domains (or ovomucoid third domain derivatives) with five serine proteinases--(alpha)-chymotrypsin, elastase I, subtilisin Carlsberg, Streptomyces griseus proteinase A and B--were measured. The techniques of making such measurements were extensively studied and improved. The analysis of the data is greatly aided by several high resolution three-dimensional structures of free ovomucoid third domains and especially of ovomucoid third domain-enzyme complexes. In interpreting the data, two major simplifying assumptions were made. The first--which was proposed earlier by Empie and Laskowski (Biochemistry 21, 2274, 1982)--is that changes in residues not in contact with the enzyme do not affect K(,a)('obs). This was generally confirmed with additional natural variants of ovomucoid third domains, but some exceptions were found. The second proposed assumption is that residue contributions are additive. It is assumed that changing residue X(,1) to X(,2) at a specified sequence position is independent of the remainder of the amino acid sequence. Eleven additivity cycles, i.e. situations where the same substitution could be observed in two different sequences, were studied. It is shown that additivity holds in most but not in all cases. In some cases, the failure of additivity assumption can be explained from the known three-dimensional structures. Eleven residues of ovomucoid third domains are in contact with enzymes in enzyme-inhibitor complexes. Of these, the P(,1) (primary reactive site) residue is the most important, but substitution at the contact positions other than P(,1) often exerts large effects on K(,a)('obs). Some of these effects are differential, which means while the interaction with one enzyme is greatly improved, the interaction with another is lessened. The tables of effects of various contact residues upon enzyme-inhibitor association allowed me to construct hypothetical ovomucoid third domains which, according to my expectation, will be highly specific for just one of the five enzymes I have studied. Future tests on artificial variants will confirm or deny these predictions.

Degree

Ph.D.

Subject Area

Biochemistry

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