Biochemical characterization of an invariant histidine involved in Escherichia coli DNA topoisomerase I catalysis.
An invariant histidine residue, His-365 in Escherichia coli DNA topoisomerase I, is located at the active site of type IA DNA topoisomerases and near the active site tyrosine. Its ability to participate in the multistep catalytic process of DNA relaxation was investigated. His-365 was mutated to alanine, arginine, asparagine, aspartate, glutamate, and glutamine to study its ability to participate in general acid/base catalysis and bind DNA. The mutants were examined for pH-dependent DNA relaxation and cleavage, salt-dependent DNA relaxation, and salt-dependent DNA binding affinity. The mutants relax DNA in a pH-dependent manner and at low salt concentrations. The pH dependence of all mutants is different from the wild type, suggesting that His-365 is responsible for the pH dependence of the enzyme. Additionally, whereas the wild type enzyme shows pH-dependent oligonucleotide cleavage, cleavage by both H365Q and H365A is pH-independent. H365Q cleaves DNA with rates similar to the wild type enzyme, whereas H365A has a slower rate of DNA cleavage than the wild type but can cleave more substrate overall. H365A also has a lower DNA binding affinity than the wild type enzyme. The binding affinity was determined at different salt concentrations, showing that the alanine mutant displaces half a charge less upon binding DNA than an inactive form of topoisomerase I. These observations indicate that His-365 participates in DNA binding and is responsible for optimal catalysis at physiological pH.
Perry
Kay
K
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208, USA.
Mondragón
Alfonso
A
D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005454:Fluorescence Polarization; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed
D017931:DNA Primers; D006639:Histidine; D004264:DNA Topoisomerases, Type I