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Title: Modeling the inhibitor activity and relative binding affinities in crude enzyme-inhibitor-protein systems: insights into developmental regulation

Author
item Fish, Wayne
item MADIHALLY, S. - OKLAHOMA STATE UNIVERSITY

Submitted to: Biotechnology Progress
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2004
Publication Date: 6/4/2004
Citation: Fish, W.W., Madihally, S.V. 2004. Modeling the inhibitor activity and relative binding affinities in crude enzyme-inhibitor-protein systems: insights into developmental regulation. Biotechnology Progress. 20(3):721-727.

Interpretive Summary: In many plant and animal systems there is a means for control of degradative processes that utilizes an inhibitor-protein to bind to- and stop the action of its target enzyme. Plants frequently employ interactions of this nature against invading disease organisms. A problem encountered in trying to study these systems and their interactions is how to quantify the level of inhibitor-protein activity. Two models were derived to allow quantification of inhibitor activity. One of the models can also be utilized to measure the strength of binding between inhibitor-protein and its target enzyme. These models were successfully employed to better define the interaction between a natural inhibitor-protein of developing cantaloupe fruit and its target enzyme from a fruit-rot pathogen. The equations derived in this investigation can be applied to a broad spectrum of plant - pathogen systems and will aid plant breeders in the development of naturally enhanced plant disease-resistance systems.

Technical Abstract: Within a number of classes of hydrolytic enzymes are certain enzymes whose activity has been shown to be modulated by a specific inhibitor-protein that binds to the enzyme and forms an inactive complex. One unit of a specific inhibitor-protein's activity is often defined as the amount necessary to inhibit one unit of its target enzyme by fifty percent. No objective quantitative means is available to determine this point of fifty percent inhibition in crude systems such as those encountered during purification. Two models were derived: the first model is based on an irreversible binding approximation, and the second, or equilibrium, model is based on reversible-binding. The two models were validated using the inhibition data for the polygalacturonase - polygalacturonase-inhibiting protein system. Results suggest that the first model can be used for inhibitor-protein activity determination and the second model can be used for inhibitor-protein activity determination as well as for comparison of association constants among enzymes and their inhibitor-proteins from multiple sources. The models were used to identify and further clarify the nature of a differential regulation of expression of polygalacturonase - inhibiting protein in developing cantaloupe fruit. Application of these models should prove valuable in gaining insights into regulatory mechanisms and enzyme - inhibitor-protein interactions.