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/

We have used two structurally well-characterized serine proteinase var iants, subtilisins Carlsberg and BPN', to produce (Cys)-S-/(His)-Im(+) H ion-pairs by chemical mutation in well defined, different, electrost atic microenvironments. These ion-pairs have been characterized by pH- dependent rapid reaction kinetics using, as reactivity probes, thiol-s pecific, time dependent inhibitors, 2,2'-dipyridyl disulfide and 4,4'- dipyrimidyl disulfide,that differ in the protonation states of their l eaving groups in acidic media, computer modelling and electrostatic po tential calculations. Both ion-pairs possess nucleophilic character, i dentified by the striking rate maxima in their reactions with 2,2'-dip yridyl 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 electro static influence other than the deprotonation of His63 (pK(a) 10.2). I n the BPN' enzyme, the analogous, (Cys221)-S-/(His64)-Im(+)H ion-pair is produced by protonic dissociation with pK(a) 5.1 and its reactivity is affected by an ionization with pK(a) 3.5 in addition to the deprot onation of His64 (pK(a) greater than or equal to 10.35). It is a strik ing result that calculations using finite difference solutions of the Poisson-Boltzmann equation provide a value of the pK(a) difference bet ween the two enzyme catalytic sites (0.97) in close agreement with the value (1.0) determined by reactivity probe kinetics when a protein di electric constant of 2 is assumed and water molecules within 5 Angstro m of the catalytic site His residue are included. The pK(a) difference is calculated to be 0.84 when the water molecules are not included an d a protein dielectric constant of 20 is assumed. The calculations als o identify Glu156 in the BPN' enzyme (which is Ser in the Carlsberg en zyme) as the main individual source of the pK(a) shift. The additional kinetically influential pK(a) of 3.5 is assigned to Glu156 by examini ng the non-covalent interactions between the 2-pyridyl disulfide react ivity probe and the enzyme active centre region. (C) 1996 Academic Pre ss Limited