An essential role for the conserved Glu-133 in the anion interaction with superoxide dismutase.
Negatively charged glutamic acid residues at positions 132 and 133 in human Cu2Zn2SOD are located at the entrance to the active site cavity and affect electrostatic interactions with the negatively charged substrate. The mutants in which these residues have been neutralized separately and together by conversion to glutamine residues or changed to a positive group on position 133 have been characterized through a variety of biophysical techniques. The structure around the metal ions, as monitored by spectroscopic measurements, is the same in the mutants and native enzyme. The mutants have been characterized with respect to the affinity for the anion N3-. The mutants have larger affinity for azide than the WT, as a result of the removal of one or two negative charges or of the introduction of a positive group. The pattern of the azide affinity constants parallels that of the rate of O2- dismutation. The substitution of the negative Glu-133 with a positive group does not induce a larger increase in the affinity as well as in the catalytic rates with respect to its neutralization. These patterns cannot, therefore, be rationalized only in terms of electrostatic interactions. The behavior of the mutants towards the substrate (O2-) and substrate analogue (N3-) is discussed on the basis of theoretical predictions available in the literature.
Banci
L
L
Department of Chemistry, University of Florence, Italy.
Cabelli
D E
DE
Getzoff
E D
ED
Hallewell
R A
RA
Viezzoli
M S
MS
D000838:Anions; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D005971:Glutamates / Q000737:chemistry*; D018698:Glutamic Acid; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000737:chemistry*
D000838:Anions; D001386:Azides; D005971:Glutamates; D018698:Glutamic Acid; D013482:Superoxide Dismutase