J. Biochem, 1967, Vol. 62, No. 3 300-307
© 1967 Japanese Biochemical Society
research-article |
Correlation between Spectral Changes and Synergistic Inhibitionof Bacterial Phosphopyruvate Hydratase in the Presence of Two Metal Ions
From Noda Instituite for Scientific Research Noda, Chiba
The combined effects of Mg++ and other activating metal ions upon the activity of bacterial phosphopyruvate hydratase [EC 4.2.1.11
[EC]
] were investigated, and synergistic inhibition was observed in the presence of both Mg++ and Zn++. This synergism was correlated with changes in the ultraviolet spectrum of the enzyme upon binding with Mg++ and Zn++. It was suggested that the enzyme possesses at least two metal-binding sites; t. e., a catalytic site and an inhibitory site. Binding of a metal ion to the catalytic site caused a decrease in absorption at about 260 mµ. The magnitude of this spectral change (
A260) was proportional to the initial velocity of the enzyme reaction, and the apparent dissociation constant for this binding agreed well with the apparent Michaelis constant for the same metal ion. Binding of a metal ion, especially Zn++, to the inhibitory site, probably a tryptophyl residue, caused an increase in absorption at about 295 mµ. This binding resulted in the inhibition of the enzyme, and the magnitude of the spectral change in this region (A295) was proportional to the degree of inhibition. The interaction between substrate and metal ions could not account for the complicated inhibition observed in the presence of both Mg++ and Zn++.