Isolation, purification, and partial characterization of an AMP deaminase from Saccharomyces cerevisiae D

Abstract

An adenosine 5'-monophosphate deaminase (AMP aminohydrolase, EC 3.5.4.6) was purified to homogeneity from the cell-free extract of Saccharomyces cerevisiae D KCTC7248. The molecular mass of subunit was estimated to be 80 kDa on SDS-PAGE, and that of the holoenzyme was shown to be 240 kDa by gel filtration. The isoelectric point of the enzyme (AMP deaminase D) was determined to be 6.2. The AMP deaminase D was specific towards AMP with an apparent K(m) value of 4.1 mM and a Hill coefficient, n(H), of 2.2. Both ATP and ADP were positive allosteric effectors of the AMP deaminase D: The apparent K(m), was decreased to 1.6 mM and 3.3 mM in the presence of 0.1 mM ATP and ADP, respectively, lowering n(H) to 1.0. Univalent cations like K+, Na+, and Li+ activated the enzyme but some divalent cations such as Cu2+ and Cd2+ showed strong inhibitory effects. This enzyme displayed optimum activity at 30°C and pH 7.0. In addition, it was stable up to 45°C and over a wide pH range (pH 5.5-9.0). Amino acid sequences of its N-terminal region were analyzed to be ADYKMQMFADDA.