Substrate-dependent kinetic behavior of horse plasma cholinesterase: evidence for kinetically distinct populations of active sites.

The study investigated the varying reactions of horse plasma cholinesterase when inhibited by propranolol, demonstrating that the reactions depend on the type of substrate used. They proposed a model where active sites in the enzyme process are distinct and can react differently depending on the substrate.

Findings

• The study found that the inhibition of horse plasma cholinesterase by propranolol, a class of drug used to treat heart conditions, varied depending on the substrate used. A substrate is a molecule acted upon by an enzyme. In this case, the two substrates tested were butyrylthiocholine and p-nitrophenylbutyrate.
• With butyrylthiocholine as a substrate, the inhibition demonstrated a first-order dependence on the concentration of the inhibitor, documented by a Ki of 8 microM.
• However, when p-nitrophenylbutyrate was used as the substrate, the researchers observed a biphasic v-1 versus [I] relationship, which means the velocity of the reaction decreases as the concentration of the inhibitor increases. This biphasic curve was broken down into two components, with noteworthy Ki’s at 9 microM and 1.3 mM.

Interpretation of Findings

• The fact that different substrates led to different types of inhibition of the plasma cholinesterase led the researchers to theorize that there might be two kinds of active sites on the enzyme, each able to process one of these substrates.
• It also resulted in partial inhibition of p-nitrophenylbutyrate hydrolysis when butyrylthiocholine was alternatively used as the substrate. Moreover, inhibition of butyrylthiocholine hydrolysis by p-nitrophenylbutyrate could be accounted for by pure competitive inhibition at two sites.
• The researchers interpreted their findings with a four-site, low-symmetry model. In this model, the enzyme possesses four active sites, two of which can process both substrates, and the other two can only process p-nitrophenylbutyrate.