Study of the electrostatic effects of mutations on the surface of dehaloperoxidase-hemoglobin A

Point mutations of dehaloperoxidase-hemoglobin A (DHP A) that affect the surface charge have been prepared to study the interaction between DHP A with its substrate 2,4,6-trichlorophenol (TCP). Kinetic studies of these surface mutations showed a correlation, in which the more positively charged mutants have increased catalytic efficiency compared with wild type DHP A. As a result, the hypothesis of this study is that there is a global electrostatic interaction between DHP A and TCP. The electrostatic nature of substrate binding was further confirmed by the result that kinetic assays of DHP A were affected by ionic strength. Furthermore, isoelectric focusing (IEF) gel study showed that the pI − 6.8 for DHP A, which indicates that DHP A has a slight negative charge pH 7, consistent with the kinetic observations.

► Surface mutations alter surface charge and affect enzymatic rate.
► Ionic strength effect proves electrostatic contribution to substrate binding interaction.
► Correlation of positive charge modification with acceleration of enzymatic rate.
► Native enzyme-substrate electrostatic contribution is slightly repulsive.
► Isoelectric focusing confirms moderate negative charge of wild type enzyme surface.