Yeast Inorganic Pyrophosphatase. Functional and ¹¹³Cd²⁺ and ³¹P Nuclear Magnetic Resonance Studies of the Cd²⁺-Enzyme

We have exploited the nuclear spins of ¹¹³Cd²⁺ and ³¹P to characterize Cd²⁺ binding sites on yeast inorganic pyrophosphatase (PPase) and to elucidate the nature of the Cd²⁺-inorganic phosphate (P_i) interaction on the enzyme surface. We have also carried out functional studies on the Cd²⁺-enzyme to test the relevance of our results for the more active Mg²⁺-enzyme. Our major results and conclusions are as follows: (1) Equilibrium dialysis (using ¹¹⁵Cd²⁺) and ¹¹³Cd NMR agree in showing the presence of one high-affinity and several low-affinity Cd²⁺ sites per enzyme subunit in the absence of P_i and three high-affinity sites per subunit in the presence of P_i. This latter result agrees with previous results found with Mg²⁺, Mn²⁺, and Co²⁺ (Springs et al., 1981; Cooperman et al., 1981). (2) By their chemical shifts, all three tightly bound ¹¹³Cd²⁺ ions in the presence of P_i have predominantly or exclusively oxygen ligands in their inner spheres. This is consistent both with chemical modification studies, indicating the absence of lysines, histidines, and cysteines, and with our inability to oxidize enzyme-bound Co²⁺ to Co³⁺, a process which would require inner-sphere nitrogen coordination to Co²⁺. (3) In the presence of three bound ¹¹³Cd²⁺ ions per subunit, we observe distinct P_i resonances corresponding to P_i bound to the high-affinity site (site 1) and to the low-affinity site (site 2). Both of these resonances show large downfield shifts from P_i in solution (∼8 ppm and ∼3 ppm, respectively). Interestingly, binding of P_i to site 2 modulates the chemical shift in site 1, causing a shift of some 0.8 ppm upfield. This modulation supports the idea that the two sites are close to one another. (4) The line width for P_i bound to site 1 narrows when ¹¹²Cd²⁺ (I = 0) replaces ¹¹³Cd²⁺ (I = ½), thus providing very strong evidence for scalar coupling and inner-sphere binding between ¹¹³Cd²⁺ and ³¹P_i. By contrast, little narrowing is seen for P_i bound to site 2, a result consistent with, but not proving, a lack of inner-sphere binding at this site. (5) Distinct ³¹P resonances corresponding to enzyme-bound P_i are only seen in the presence of three bound Cd²⁺ ions per subunit, despite the fact that P_i is bound to PPase even in the absence of Cd²⁺. This observation is consistent with other evidence for a conformational change accompanying the binding of a third metal ion per subunit. (6) Measures of catalytic function (PP_i hydrolysis, H₂O-P_i oxygen exchange, enzyme-bound PP_i formation) obtained in the presence of Cd²⁺ allow calculation of the equilibrium constants and rate constants for overall PPase catalysis. The major result is that whereas the affinities for PP_i and both P_i bindings are not very different from the values found in the presence of Mg²⁺ (constants within an order of magnitude), the rate constants corresponding to PP_i hydrolysis and re-formation on the enzyme are drastically lower, ∼ 10⁵-fold. (7) Because of the slow rate constants involved, the pre-steady-state rate of EPP_i formation could be measured without need of a rapid quench apparatus. The rate constant obtained agrees within experimental error with that predicted from the values of k₃ and k₄ obtained from equilibrium and steady-state measurements, demonstrating the adequacy of our minimal scheme in accounting for PPase catalysis, at least in the presence of Cd²⁺.