Carboxamidomethylation of Yeast Inorganic Pyrophosphatase

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Abstract
1) Yeast inorganic pyrophosphatase [EC 3.6.1.1, PPiase] was found to be inactivated by iodoacetamide. 2) The rate of inactivation of PP,ase by iodoacetamide was minimum at pH 7.0 in the range tested. 3) When PPiase was inactivated by iodoacetamide at pH 5.5 and 8.0, it was found that about one and three carboxamidomethyl groups were incorporated into PPiase, respectively. 4) The amino acid residue of PPiase modified by this reagent at pH 5.5 was identified as methionine by paper chromatography and paper electrophoresis of the hydrolysate of 14C-iodoacetamide-modified enzyme. 5) The amino acid residues modified by iodoacetamide at pH 8.0 were identified as methionine, lysine, histidine, and cysteine. The molar ratio of these modified amino acids was 1.2, 0.9, 0.7, and 0.2. 6) Fluorescence emission spectrum, spectrophotometric titration of phenolic groups, N-bromosuccinimide (NBS) oxidation of tryptophan residues and solvent perturbation difference spectrum of carboxamidomethylated PPiases were studied, and it was concluded that the environment of the tryptophan residues in modified PPiase differs markedly from that in native PPiase. 7) On the basis of the above experiments, the importance of the methionine residue for the enzymatic activity of PPiase was deduced and the possible role of the methionine residue in PPiase activity was also discussed.

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