Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. Evidence for a dinuclear zinc site.
XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom. The data indicate that zinc is not required for HPRG/AMPD interaction, both zinc ions being bound to the catalytic subunit of the enzyme, one to the three conserved amino acid residues among those four assumed to be in contact with zinc in yeast AMPD, and the other at the N-terminal region, probably to His-52, Glu-53 and His-57. Tryptic digests of different enzyme preparations demonstrate the existence of two different protein conformations and of a zinc ion connecting the N-terminal and C-terminal regions of AMPD.
Mangani
Stefano
S
Dipartimento di Chimica, Università di Siena, Via Aldo Moro, 53100-Siena, Italy; CERM, Università di Firenze, Via Luigi Sacconi 6, 50019 Firenze, Italy.
Benvenuti
Manuela
M
Moir
Arthur J G
AJ
Ranieri-Raggi
Maria
M
Martini
Daniela
D
Sabbatini
Antonietta R M
AR
Raggi
Antonio
A
D000659:AMP Deaminase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D005583:Fourier Analysis; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology*; D011817:Rabbits; D051381:Rats; D013057:Spectrum Analysis / Q000379:methods; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D000659:AMP Deaminase; D015032:Zinc