X-ray crystallographic and nuclear magnetic resonance spectral studies of the products from the yeast inorganic pyrophosphatase-Co(NH3)4PP reaction. Investigation of the pyrophosphatase reaction mechanism

Abstract

Yeast inorganic pyrophosphatase catalyzes the hydrolysis of P1,P2-bidentate Co(NH3)4 pyrophosphate [Co(NH3)4PP] to the cis, bis(phosphate) complex Co(NH3)4(Pi)2, which is not stable at neutral pH and over a period of 24 h converts to HPO4(2-) and a mixture of bidentate Co(NH3)4(PO4) and monodentate Co(NH3)4(H2O)(HPO4). Concurrent with this process is the reduction and subsequent release of Co(H2O)6(2+) from the cobalt tetraammine bis(phosphate) complex and/or the cobalt tetraammine monophosphate complex. Bidentate tetraammine phosphatocobalt (III), hexaaquocobalt(II), orthophosphate, and two free water molecules cocrystallize [Co(NH3)4PO4 . Co(H2O)6(2+) . HPO4(2-) . 2H2O] from the reaction mixture in the triclinic space group Pi (Z = 2) with cell dimensions a = 6.849 (1) A, b = 11.693 (2) A, c = 12.630 (2) A, alpha = 65.60 (1) degree, beta = 88.98 (1) degree, and gamma = 73.04 (1) degree. The structure was solved by the heavy atom technique and refined to an R index of 0.040 by using 3077 intensities measured up to a 2 theta limit of 155 degrees. 31P NMR studies of the equilibrium mixture reveal that the equilibrium constant is a sensitive function of solution pH and temperature. Unlike the Co(NH3)4PP complex, there is evidence indicating that the Mg(H2O)4PP complex is degraded to monodentate Mg(H2-O)5PO4 in the enzyme active site.

Investigation of the regiospecificity and stereospecificity of proton transfer in the yeast inorganic pyrophosphate catalyzed reaction

“…Thus, we predicted that PPase could catalyze the hydrolysis of the bidentate metal-PNP complex if and only if it could transfer a proton to the nitrogen atom during or prior to catalysis. Previous studies (Knight et al, 1981;Haromy et al, 1982Haromy et al, , 1983 have shown that (1) MPNP is a tight competitive inhibitor vs. MPP (and hence that is binds to the PPase substrate binding site), (2) P',P2-bidentate Co(NH3)4PP¡ is a substrate for PPase, (3) P',P2-bidentate Co(NH3)4PNP and P',P2-bidentate Co(NH3)4PP¡ are isosteric, and (4) phosphoryl transferring enzymes that mediate proton transfer to the oxygen atom undergoing bond cleavage can do so to the nitrogen atom of an imidophosphate substrate analogue. In this study we found that PPase was unable to catalyze the hydrolysis of MgPNP and P',P2-bidentate Co(NH3)4PNP under conditions that lead to rapid conversion of MgPP¡ and P',P2-bidentate Co(NH3)4PP¡.…”

“…Thus, in order to generate stable stereochemical probes for our studies, the exchange-inert metal ions Cr(III) and Co(III) were used in place of the Mg(II) ion to form the metal-PPS complex. Both P1,P2-bidentate Cr(III)PP¡ and Co(III)PP¡ have been previously shown to be substrates for PPase (Knight et al, 1981;Haromy et al, 1982), and it was expected that the Cr(III) and Co(III) complexes of PPS would likewise be substrates.…”

“…Previous studies in our laboratory have identified the active substrate in the PPase-catalyzed reaction as the P1,P2-bidentate Mg(II) complex of the pyrophosphate tetraanion (Knight et al, 1981(Knight et al, , 1983. During the initial catalytic step, the MgPP¡ complex is cleaved to the rij-[Mg(P¡)2] complex (Haromy et al, 1982). In the absence of enzyme-mediated proton transfer to the phosphoryl group that is displaced during the course of the hydrolytic cleavage of MgPPj2", Mg(II)-coordinated phosphate trianion would be generated.…”

Biochemistry of the Elemental Halogens and Inorganic Halides

“…Other mechanistic studies have demonstrated that the active substrate in the pyrophosphatase-catalyzed reaction is the bidentate Mg2+ complex of the pyrophosphate tetraanion MgPPj • Enzyme active site-mediated proton transfer converts a phosphoryl group into a good leaving group and Mg(PJ2 is produced (Haromy et al, 1982) by hydrolytic cleavage prior to, or synchronous with, proton transfer (Lin et al, 1986;Knight et aI., 1983Knight et aI., , 1981 (Fig. 2-6).…”

A Structural Model for HPO42− Binding to Co in a Water Oxidation Catalyst

“…The observation of three peaks in the aliphatic region of the 13 C NMR spectrum of 1 was consistent with exclusive formation of the 6 geometric isomer;18, 19 such a preference has been observed previously in complexes containing the pmea ligand. A single peak at 20.2 ppm in the 31 P NMR spectrum of 1 confirms chelation, rather than monodentate coordination, of the HPO 4 2− ligand in D 2 O solution,20 while the 59 Co NMR chemical shift of 9064 ppm is slightly greater than that of [(pmea)Co(O 2 CO)] + , indicating that HPO 4 2− is a weaker‐field ligand than CO 3 2− .…”