Role of Metals in the Class II Aldolases. Spectral Studies of Cobalt Yeast Aldolase

The binding of 2 moles of cobalt(II) to each mole of yeast apoaldolase produces an active enzyme, exhibiting an absorption spectrum with maxima at 535 (155), 485 (125), 1290 (18), and 1640 mμ (18 M^-1 cm^-1). The visible absorption bands are optically active, with circular dichroic bands of molecular ellipticities -2800 and -2500 (deg cm²) per dmole, respectively. The addition of saturating concentrations of the substrate, fructose 1,6-diphosphate, results both in hypso- and hypochromic shifts of the main absorption band in the visible region. In contrast, neither K⁺, an activator, nor phosphate ion, an inhibitor, affect these spectral properties of the cobalt enzyme. The optical asymmetry of the active-site cobalt atoms seems closely related to enzymatic activity, as is demonstrated by its correlation to the changes of the circular dichroic spectrum on titration to pH values of less than 8. The present studies are discussed in the context of the proposal that the metal atom at the active site of yeast aldolase serves as an electrophile, aiding in bond polarization to facilitate the aldolytic cleavage of the substrate.