(622v) Analysis of Kinetics of a Serpin-Serine Protease Complex

Alpha-1-Antitrypsin (A1AT) Deficiency (A1AD) is a potentially debilitating inherited condition.  It is associated with reduced levels of active A1AT being secreted from the liver thus resulting in an imbalance between A1AT and Human Neutrophil Elastase (HNE) in the lungs and predisposing those affected to early-onset emphysema. Further, certain genetic mutations lead to an accumulation of aberrant A1AT in the liver potentially leading to cirrhosis. No cure exists, and current approaches for diagnosis are insufficient as illustrated by the fact that A1AD is underdiagnosed and diagnosis is often delayed. In this work, a synthetic substrate (N-Succinyl-Ala-Ala-Ala-p-Nitroanilide), an enzyme-inhibitor system consisting of Porcine Pancreatic Elastase (PPE, an often-used surrogate for HNE) and a “normal” form of A1AT, and a mathematical model based on irreversible inhibition are used to understand rates of product formation and modes of inhibition. Tian et al. [Biochemistry, 21,1982] used basic conservation equations previously developed, with a modification for irreversibility, to describe enzyme inhibition for complexes traveling down an irreversible pathway but in which a reversible complex (e.g., Michaelis-type) is formed along the way.  That method goes on to describe approaches for exploring whether such inhibitors behave  in competitive, non-competitive, or an uncompetitive manner, and the approaches reported there are used herein to characterize the A1AT-PPE system.  The physiologic level of A1AT in the body varies between and within individuals and is associated with, but not totally diagnostic for, the development of lung and/or liver complications. It has been shown in this work that such modeling could be a useful tool to describe the interaction between A1AT and PPE, and as such, be important for the development of new diagnostic approaches for A1AD.