Studies on the Tryptophan Residues of Yeast Inorganic Pyrophosphatase in Relation to the Enzymatic Activity

Abstract

In order to investigate the role of tryptophan residues on the activity of inorganic pyrophosphatase [EC 3.6.1.1, PP_iase], several lines of experiments were carried out. Methylenediphosphonate (PCP) was found to be a competitive inhibitor for PP_iase and its K_i value was found to be 1.1×10⁻³ M at pH 6.0. The ORD curve of PP_iase-Mg₂₊PCP complex showed the decrease of the depth of trough at 233 mμ and apparent change of the Cotton effect around 290 mμ in shape comparing with the ORD curve of PP_iase. The CD curve of PP_iase-Mg²⁺PCP complex showed a slight blue shift of negative CD band around 290 mμ from that of PP_iase. The difference spectrum between PP_iase and PP_iase bound with Mg²⁺PCP had a negative peak at 300 mμ indicating the possible participation of tryptophan residues for enzyme-inhibitor complex. These results suggested that the environment around some tryptophan residues was changed by the binding with Mg²⁺PCP. About one mole of tryptophan residue of PP_iase was oxidized by NBS at pH 6.0–6.5 with simultaneous loss of the enzymatic activity. The rate of NBS oxidation was suppressed by the presence of Mg²⁺PCP. PP_iase which was oxidized with NBS lost its Cotton effect around 290 mμ. These experiments described above showed that the tryptophan residue has an important role on the enzymatic activity. The fluorescence emission spectrum of PP_iase excited by 295 mμ light was found to have maximum at 330 mμ and the maximum was shifted to 350 mμ in 6M guanidine-HCl. The pH dependence of fluorescence emission maximum indicated that at pH 6.0–7.5 where most of our experiments were carried out, the amount of tryptophan residues exposed to the solvent was minimum. Solvent perturbation difference spectra of PP_iase in the presence and absence of Mg²⁺PCP using ethylene glycol as a perturbant, gave two peaks at 285 mμ and 278 mμ and a shoulder at 295 mμ. By comparing Δε at peak positions with those of N-acetyltyrosinamide and N-acetyltryptophan, it was suggested that about 10 residues of tyrosine and 2.5 residues of tryptophan were exposed to the solvent in the absence of Mg²⁺PCP, and that 5 residues of tyrosine and 1.4 residues of tryptophan were exposed in the presence of Mg²⁺PCP.