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 adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various 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 coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound 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 site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 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 Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.
Conyers
G B
GB
Departments of Biology and Biophysics and the McCollum-Pratt Institute, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Wu
G
G
Bessman
M J
MJ
Mildvan
A S
AS
D017766:Acid Anhydride Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001473:Bartonella / Q000201:enzymology*; D001667:Binding, Competitive; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D013379:Substrate Specificity
D002413:Cations, Divalent; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D008345:Manganese; D017766:Acid Anhydride Hydrolases; C036849:bis(5'-nucleosyl)tetraphosphatase (symmetrical); D008274:Magnesium; D002118:Calcium