Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: Magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adeno sine 5'-tetraphospho-5'-adenosine (Ap(4)A) pyrophosphatase of 170 amino aci ds that catalyzes the hydrolysis of Ap(4)A, Ap(5)A, and Ap(6)A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cart wright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When var ious divalent cations were tested over a 300-fold concentration range, Mg2, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not . Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill co efficients of 3.0 and 1.6 and K-0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+. Ap(4)A showed hyperbolic kinetics with K-m values of 0. 34 mM: for both Mn2+. Ap(4)A and Mg2+. Ap(4)A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the long itudinal relaxation rate of water protons revealed two Mn2+ binding sites p er molecule of Ap(4)A pyrophosphatase with dissociation constants of 1.1 mM , comparable to the kinetically determined K-0.5 value of Mn2+. The enhance ment factor of the longitudinal relaxation rate of water protons due to bou nd Mn2+ (epsilon(b)) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating si te-site interaction between the two enzyme-bound Mn2+ ions, Assuming the de crease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 Angstrom between them . The substrate Ap(4)A binds one Mn2+ (K-d = 0.43 mM) with an epsilon(b) va lue of 2.6, consistent with the molecular weight of the Mn2+. Ap(4)A comple x. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with K-d values of 8.6 mM and one Mg2+ binding site on Ap(4)A with a K-d Of 3.9 mM, values that are comparable to the K-0.5 for Mg 2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites we re found - two on the enzyme and one on the substrate - with dissociation c onstants comparable to the kinetically determined K-0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activat e Ap(4)A pyrophosphatase but inhibits the Mn(2+-)activated enzyme competiti vely with a K-i = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/ - 1.3 mM) and one on Ap(4)A with a dissociation constant of 2.1 mM. These v alues are similar to its K-i suggesting that inhibition by Ca2+ results fro m the complete displacement of Mn2+ from the active site. Unlike the homolo gous MutT pyrophosphohydrolase, which requires only one enzyme-bound divale nt cation in an E . M2+. NTP . M2+ complex for catalytic activity, Ap(4)A p yrophosphatase requires two enzyme-bound divalent cations that function in an active E .(M2+)(2). Ap(4)A . M2+ complex.