Cooperativity of papain-substrate interaction energies in the S2 to S2' subsites.
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 kinetic constants. The observed specificity energies [defined as delta delta G obs = -RT ln [(kcat/KM)first/(kcat/KM)second)]] of the substrate backbone hydrogen bonds were -2.7 kcal/mol for the P2 NH and -2.6 kcal/mol for the P1 NH when compared against substrates containing esters at those sites. The observed binding energies were -4.0 kcal/mol for the P2 Phe side chain, -1.0 kcal/mol for the P1' C=O, and -2.3 kcal/mol for the P2' NH. The latter three values probably all significantly underestimate the incremental binding energies. The P2 NH, P2 Phe side-chain, and P1 NH contacts display a strong interdependence, or cooperativity, of interaction energies that is characteristic of enzyme-substrate interactions. This interdependence arises largely from the entropic cost of forming the enzyme-substrate transition state. As favorable contacts are added successively to a substrate, the entropic penalty associated with each decreases and the free energy expressed approaches the incremental interaction energy. This is the first report of a graded cooperative effect. Elucidation of favorable enzyme-substrate contacts remote from the catalytic site will assist in the design of highly specific cysteine protease inhibitors.
Berti
P J
PJ
Protein Engineering Section, Biotechnology Research Institute, Montréal, Québec, Canada.
Faerman
C H
CH
Storer
A C
AC
D000494:Allosteric Regulation; D001665:Binding Sites; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D010206:Papain / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D010206:Papain