Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. A new model for substrate interactions at a dinuclear cocatalytic Zn site

The Role of Histidine-Proline-Rich Glycoprotein as Zinc Chaperone for Skeletal Muscle AMP Deaminase

“…This is consistent both with the presence of specific interactions between the N -terminal and C -terminal regions of rabbit skeletal muscle AMPD as suggested by the structural role that can be played by zinc in stabilizing one among the possible enzyme conformations and with the presence in the HPRG component of a disulfide bridge connecting the N -terminal and C -terminal regions (Figure 3). The observation that the tryptic peptides derived from the N -terminal region of HPRG are liberated in constant association with fragments derived from the AMPD N -terminus suggests that the N -terminus of HPRG should be exposed as well as the AMPD region that encompasses the regulatory domain associated with the peculiar inhibition of the enzyme by ATP [41,50,51] and strengthens the hypothesis of a physiological role of HPRG in preserving the molecular integrity of the enzyme. The protective role of HPRG against the fragmentation of AMPD by thiol proteases is suggested by the presence of two modules homologous to cystatin (1–112, 113–229) at the N -terminus of the protein (Figure 3) and by the observation that the rate of the fragmentation of HPRG-enriched AMPD on storage is reduced [10].…”

“…Moreover, X-ray absorption spectroscopy of Zn-peptide binary and ternary complexes prepared using a number of synthetic peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMPD strongly suggested that the region 48–61 of the enzyme contains a zinc binding site whilst region 360–372 is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD [49]. X-ray absorption spectroscopy performed on fresh preparations of rabbit skeletal muscle AMPD provided evidence for a dinuclear zinc site in the enzyme compatible with a (µ-aqua)(µ-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn–Zn distance of about 3.3 Å [50]. The data also indicated that two zinc ions are 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-51, Glu-53 and His-57 (Figure 1A).…”

“…( A ) A model of dinuclear Zn site in rabbit skeletal muscle AMP deaminase (AMPD) [49]; ( B ) Proposed interactions of the substrate at the dinuclear cocatalytic Zn site of rabbit skeletal muscle AMPD [50]. The amino acid residues involved in the binding in the catalytic Zn 1 region are based on the proposed interactions of the substrate transition state at the catalytic site of adenosine deaminase [47] and amino acid homology to rabbit AMPD1.…”

Structure-function relationships in mammalian histidine-proline-rich glycoprotein

“…X-ray absorption spectroscopy of freshly prepared rabbit skeletal muscle AMPD indicated the presence of a dinuclear zinc site fitting with a (m-aqua) (m-carboxylato) dizinc(II) core, with two histidines at each metal site and a ZneZn distance of about 3.3 Å [83]. The two Zn 2þ ions in the AMPD metallocenter play different roles in the catalysis, one of them (Zn1) could polarize the nucleophile water molecule and the other (Zn2) could transiently bind an activating AMP molecule [84]. The hypothesis that HPRG behaves as a zinc metallochaperone in the AMPD-HPRG complex is strengthened by the sequence comparative results reported in this review showing that Fig.…”

X-Ray Absorption Spectroscopy of Dinuclear Metallohydrolases

“…Rather than attempt to review all cases that have been examined by XAS, we will restrict the following discussion to just a few in which XAS has made the most significant contributions. XAS has been used to study several aminopeptidases (N-terminal protein degradation) (43,44), AMP deaminase (nucleic acid metabolism) (45)(46)(47), prolidase (iminopeptidase) (48), DapE (bacterial peptidoglycan synthesis) (49), arginase (part of the urea cycle) (38,50), the acyl-homoserinelactone (AHL) lactonase (bacterial quorum sensing) (51,52), ZiPD (an RNA processing enzyme with phosphodiesterase activity) (53)(54)(55)(56), and glyoxalase (detoxification of a-ketoaldehydes) (57,58). However, for the sake of brevity, we will focus our review on the use of XAS to study purple acid phosphatases (PAPs) and metallo-b-lactamases (MbLs).…”