Secondary enzyme-substrate contacts in papain catalyzed ester hydrolysis.

Abstract

Secondary enzyme-substrate contacts in the hydrolysis of ester substrates by the cysteine protease papain were investigated by systematically altering backbone hydrogen bonding and side chain hydrophobic contacts in the substrate and determining each substrate's $k\sb{\rm cat}/K\sb{\rm M}$. $k\sb{\rm cat}/K\sb{\rm M}$, the second order rate constant, directly reflects the equilibrium of free enzyme plus free substrate to the enzyme-substrate transition state. The incremental binding energies of the substrate backbone hydrogen bonds are estimated at $-$2.3 kcal/mol for the P$\sb2$ NH, $-$2.2 kcal/mol for the P$\sb1$ NH, and $-$2.3 kcal/mol for the P$\sb{2\sp\prime}$ NH. The observed hydrogen bonding energy for the substrate backbone P$\sb{1\sp\prime}$ C=O is $-$1.0 kcal/mol. The incremental binding energy of the Phe side chain in the P$\sb2$ position is estimated at $-$4.0 kcal/mol. The contacts on the acyl side of the scissile bond (the P$\sb2$ NH, P$\sb2$ Phe side chain and P$\sb1$ NH) display a strong interdependence of binding energies that is characteristic of enzyme-substrate interactions. This interdependence arises largely from the entropic cost of forming the rate-determining enzyme-substrate transition state. As favourable contacts are added successively to a substrate, the entropic penalty associated with each decreases and the binding energy expressed approaches the incremental binding energy. On the leaving group side, the observed $-$1.0 kcal/mol for the P$\sb{1\sp\prime}$ C=O probably underestimates the incremental binding energy whilst the $-$2.3 kcal/mol for the P$\sb{2\sp\prime}$ NH is a reasonable estimate. Elucidation of favourable enzyme-substrate contacts remote from the catalytic site will assist in the design of highly specific cysteine protease inhibitors.