EVIDENCE FOR AN ESSENTIAL HISTIDINE RESIDUE ON ACTIVE-SITE OF HUMAN URINARY DNASE-I - CARBOXYMETHYLATION AND CARBETHOXYLATION

Human urinary DNase I was inactivated by monoiodoacetate and monobromo acetate. The inactivation was greater at pH 7.2 than at 6.0 and procee ded in the presence of Ca2+. Amino acid analysis of monobromoacetate-i nactivated human urinary DNase I indicated that one histidine residue per mole of the enzyme reacted with monobromoacetate. Diethylpyrocarbo nate also inactivated the enzyme, which was protected by DNA in the pr esence of Mg2+. However, oligonucleotides did not prevent the inactiva tion even in the presence of Mg2+ Hydroxylamine almost completely rest ored the activity of the inactivated enzyme by DEP. One histidine resi due per mole of the enzyme was calculated to be modified, as shown by the difference spectra of DEP-inactivated enzyme. This histidine resid ue seems to react with the substrate. These results provide evidence t hat human urinary DNase I possesses one essential histidine residue at the active site. (C) 1994 Academic Press, Inc.