Regulation of skeletal muscle AMP deaminase. Carbethoxylation of His-51 belonging to the zinc coordination sphere of the rabbit enzyme promotes its desensitization towards the inhibition by ATP

Abstract

Background: Skeletal muscle AMP deaminase (AMPD1) regulates the concentration of adenine nucleotides during muscle contraction. We previously provided evidence that rabbit AMPD1 is composed by two HPRG 73 kDa subunits and two 85 kDa catalytic subunits with a dinuclear zinc site with an average of two histidine residues at each metal site. AMPD1 is mainly expressed in fast twitching fibers and is inhibited by ATP. The limited trypsinization of the 95-residue N-terminal domain of rabbit AMPD1 desensitizes the enzyme towards ATP inhibition at the optimal pH 6.5, but not at pH 7.1.

Methods: The modified residues of rabbit AMPD1 after incubation with radioactive diethyl pyrocarbonate ([¹⁴C]DEP) causing the desensitization to inhibition by ATP at pH 7.1 have been identified by sequence analysis and MS analysis of the radioactive peptides liberated from the carbethoxylated enzyme by limited proteolysis with trypsin.

Results: The study confirms the presence of a dinuclear zinc site in rabbit AMPD1 and shows that carbethoxylation of His-51 at the N-terminus of the catalytic subunit removes the inhibition of the enzyme by ATP at pH 7.1.

Conclusions: The desensitization to ATP is due to the modification of His-51 of the Zn₂ coordination sphere which is transduced in a conformational change of the enzyme C-terminus, where an ATP-binding site has been localized.

General significance: The progress in the study of the complex regulation of rabbit AMPD1 that shares an identical amino acid sequence with the human enzyme is important in relation to the role of the enzyme during mammalian evolution.

Keywords: AMP deaminase; AMPD1; Diethylpyrocarbonate; HPRG; Histidine-proline-rich glycoprotein; Zinc-binding motif.