Kinetic studies on ovomucoid inhibitors

Keith Donald Forwith, Purdue University

Abstract

The kinetics of ovomucoid inhibition were studied for five serine proteases, (chymotrypsin, porcine pancreatic elastase, subtilisin Carlsberg, and Streptomyces griseus proteinases A and B). It was the objective of this work to add to this wealth of inhibition information kinetic data. The primary means of assessing kinetic behavior was through the measurement of the association rate constant, k$\sb{\rm on}.$ The majority of this work was done on the third domain of ovomucoid (OM3), which can be enzymatically cleaved and purified from entire ovomucoid (OM). k$\sb{\rm on}$ was measured for 191 enzyme-OM3 pairs, 100 enzyme-OM pairs, and 10 enzyme-OM3* (modified inhibitor which has the reactive site bond cleaved) pairs. Changes in inhibitory function due to an amino acid substitution primarily affect the dissociation rate constant, k$\sb{\rm off}.$ The association rate constant remains essentially constant, at approximately $10\sp6{\rm M}\sp{-1}{\rm s}\sp{-1}.$ Steric hindrance, examined through the introduction of $\beta$-branching, can affect association rate constants, but the largest observed effect is a modest 2-3 fold decrease. Charge changes can exert larger effects. Substitution of Arg or Lys for Leu$\sp{18}$ in the primary specificity site produced a 20 fold decrease in k$\sb{\rm on}$ for subtilisin and SGPA. A charge change at P$\sb3\sp\prime$ from Met to Arg accounted for a five fold rate increase with chymotrypsin. The Arg residue forms a water mediated ion pair with Asp$\sp{64}$ of the enzyme. This specific interaction is not present in the inhibitor interactions with the other enzymes studied. Inhibition of OM3 was studied while still a part of OM. The results showed a decrease in association rate constants similar to that caused by introduction of $\beta$-branching. Individual effects on inhibition, such as the interaction of P$\sb3\sp\prime$, with chymotrypsin, were maintained in OM. Finally, the interaction of OM3* was investigated for OMTKY3* and six serine proteases, as well as the interaction of subtilisin with several OM3* variants. For single residue substitutions, a modified inhibitor demonstrated the largest effect on association rate constants, a 200 fold decrease for Arg replacement of Thr$\sp{17}$ (P$\sb2$ position). Variants which display the same thermodynamic characteristics were shown to have differing kinetic behavior.

Degree

Ph.D.

Advisors

Laskowski, Purdue University.

Subject Area

Biochemistry

COinS