Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. Evidence for a dinuclear zinc site

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

“…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). Justification for a structurally bridged dinuclear metallocenter in rabbit skeletal muscle AMPD was further supported by N -terminal analysis of the peptides liberated by limited tryptic digestion of different enzyme preparations suggesting the existence of two different protein conformations which is consistent with the hypothesis of the presence of a zinc ion connecting the N -terminal and C -terminal regions of AMPD [49]. When the limited proteolysis affects only the N -terminal 1–95 residue region of the enzyme, the consequence on the constitution of the enzyme metallocenter is probably the loss of the putative Zn 2 ion without any major change in the conformation of the remaining part of the enzyme.…”

“…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].…”

“…When the limited proteolysis affects only the N -terminal 1–95 residue region of the enzyme, the consequence on the constitution of the enzyme metallocenter is probably the loss of the putative Zn 2 ion without any major change in the conformation of the remaining part of the enzyme. In contrast, when the digestion affects the binding region of the Zn 1 ion at the C -terminus, profound changes within the AMPD structure ensue as suggested by the observed fragmentation in the middle- and C -terminal regions of the enzyme, whilst in contrast, the 58–95 region appears to be unaffected [49]. The hypothesis of a conformational change of AMPD induced or stabilized by zinc binding to the 51–60-residue region (HHEMQAHILH) is strengthened by the observation that carbethoxylation by diethyl pyrocarbonate of one or two histidine residues per subunit results in conversion of rabbit skeletal muscle AMPD into a species that, unlike the native enzyme, is not sensitive to regulation by ATP at optimal pH 6.5 [51].…”

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].…”

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).…”