Proton NMR spectroscopy as a probe of dinuclear copper(II) active sites in metalloproteins. Characterization of the hyperactive copper(II)-substituted aminopeptidase from Aeromonas proteolytica
Holz RC,
Bennett B, and Chen GJ,
Department of Chemistry and Biochemistry
Utah State University
Logan, UT 84322
Ming LJ
Department of Chemistry and
Institute for Biomolecular Science
University of South Florida
Tampa, FL 33620-5250
Abstract:
Proton NMR spectra of the hyperactive Cu(II)-substituted aminopeptidase
from Aeromonas proteolytica (AAP) were recorded in both H2O and D2O buffered
solution at pH 6.7. Several remarkably sharp, well resolved hyperfine shifted
LH NMR signals were observed in the 70 to -20 ppm chemical shift range.
That hyperfine shifted signals were observed is due to spin-coupling of
the two Cu(II) ions. Comparison of the spectra recorded in H2O and D2O
buffered solutions indicated that the signals at 44.6, 43.3, and 17.7 ppm
were solvent exchangeable. The two most strongly downfield shifted signals
were assigned to imidazole N-H protons of the two coordinated histidine
residues, while the remaining exchangeable signal was assigned to a peptidyl
N-H proton that is in close proximity to the dicopper(II) center. One-dimensional
NOE studies at pH 6.7 revealed two Y-CH2-CH< moieties that were assigned
to coordinated aspartic acid and histidine residues. In addition, a Y-CH2-CH2-CH<
moiety was also identified and was assigned to the coordinated glutamic
acid residue, Glu152. All of the hyperfine shifted signals for [CuCu(AAP)]
sharpened and shifted toward the diamagnetic region as the temperature
was increased following Curie behavior. Fits of these data and those of
a series of magnetically diverse mu-phenoxo and mu-alkoxo dicopper(II)
model complexes to the population distribution of the ground and first
excited states, provided information on the magnetic properties of-dicopper(II)
clusters. These fits indicated that the two Cu(II) ions in AAP are ferromagnetically
coupled with a 2J value of 50 + 40 cm(-1). These data provide the first
structural information regarding the hyperactive [CuCu(AAP)] enzyme and
are discussed in terms of the previously proposed mechanism of action for
AAP.
(Read an introduction about aminopeptidase, or about the metalloendopeptidase astacin)
(Read abstract about metal binding to Streptomyces AP)
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