Characterization of the Electrostatic Perturbation of a Catalytic Site (Cys)-S–/(His)-Im+H Ion-pair in One Type of Serine Proteinase Architecture by Kinetic and Computational Studies on Chemically Mutated Subtilisin Variants

Abstract We have used two structurally well-characterized serine proteinase variants, subtilisins Carlsberg and BPN′, to produce (Cys)-S − /(His)-Im + H ion-pairs by chemical mutation in well defined, different, electrostatic microenvironments. These ion-pairs have been characterized by pH-dependent rapid reaction kinetics using, as reactivity probes, thiol-specific, time dependent inhibitors, 2,2′-dipyridyl disulfide and 4,4′-dipyrimidyl disulfide, that differ in the protonation states of their leaving groups in acidic media, computer modelling and electrostatic potential calculations. Both ion-pairs possess nucleophilic character, identified by the striking rate maxima in their reactions with 2,2′-dipyridyl disulfide in acid media. In the Carlsberg enzyme, the (Cys220)-S − /(His63)-Im + H ion-pair is produced by protonic dissociation with p K a 4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (p K a 10.2). In the BPN′ enzyme, the analogous, (Cys221)-S − /(His64)-Im + H ion-pair is produced by protonic dissociation with p K a 5.1 and its reactivity is affected by an ionization with p K a 3.5 in addition to the deprotonation of His64 (p K a ≥10.35). It is a striking result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the p K a difference between the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein dielectric constant of 2 is assumed and water molecules within 5 A of the catalytic site His residue are included. The p K a difference is calculated to be 0.84 when the water molecules are not included and a protein dielectric constant of 20 is assumed. The calculations also identify Glu156 in the BPN′ enzyme (which is Ser in the Carlsberg enzyme) as the main individual source of the p K a shift. The additional kinetically influential p K a of 3.5 is assigned to Glu156 by examining the non-covalent interactions between the 2-pyridyl disulfide reactivity probe and the enzyme active centre region.