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 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 reac...

Full description

Saved in:
Bibliographic Details
Published in:Journal of molecular biology 1996-04, Vol.257 (5), p.1088-1111
Main Authors: Plou, Francisco J., Kowlessur, Devanand, Malthouse, Paul G.J., Mellor, Geoffrey W., Hartshorn, Michael J., Pinitglang, Surapong, Patel, Hasu, Topham, Christopher M., Thomas, Emrys W., Verma, Chandra, Brocklehurst, Keith
Format: Article
Language:English
Subjects:
2,2'-Dipyridyl - analogs & derivatives
2,2'-Dipyridyl - metabolism
Bacillus subtilis - enzymology
Binding Sites
Computer Simulation
Cysteine - metabolism
Disulfides - metabolism
electrostatic potential calculations
energy minimization
Histidine - metabolism
Hydrogen-Ion Concentration
Kinetics
Models, Molecular
Molecular Structure
Mutagenesis
Protein Conformation
Pyrimidines - metabolism
reactivity probe kinetics
Subtilisins - chemistry
Subtilisins - metabolism
Sulfhydryl Compounds - metabolism
Sulfhydryl Reagents - metabolism
thiolate-imidazolium ion-pairs
thiolsubtilisin variants
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 a4.1 and its reactivity is not perturbed by any detectable electrostatic influence other than the deprotonation of His63 (p K a10.2). In the BPN′ enzyme, the analogous, (Cys221)-S −/(His64)-Im +H ion-pair is produced by protonic dissociation with p K a5.1 and its reactivity is affected by an ionization with p K a3.5 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 adifference 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 Å of the catalytic site His residue are included. The p K adifference 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 ashift. The additional kinetically influential p K aof 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.
ISSN:0022-2836
1089-8638
DOI:10.1006/jmbi.1996.0225