On the state of aromatic amino acids in inorganic pyrophosphatase from bakers' yeast

Heitmann, P; Möllerke, Ch.; Uhlig, Helmut

doi:10.1515/9783112575024-005

Cited by 2 publications

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“…This need arises because the true substrate for PPase is a Mg2+ complex of P207'h We should point out that since the microscopic steps for PP¡ hydrolysis and the release of each of the P¡'s are all partially rate determining (Springs et al, 1981), proton donation from R,OH to the phosphoryl group in site 1 need not be simultaneous with water attack on the phosphoryl group in site 2. On the basis of the available evidence from chemical modification studies (Heitmann et al, 1972;Chiu, 1974;Heitmann & Uhlig, 1974) which exclude essential lysine, cysteine, or histidine residues, a tyrosine residue or a bound water molecule is the most likely candidate for RjOH and several tyrosine residues (numbers 88, 92, and 191 in the sequence) have been placed at or near the active site by Kuranova et al (1983). Complexation of R,OH to either or both of the metal ions bound to P¡ in site 1, indicated as a possibility in Figure 7, could lower the pKa of R^H to 8.…”

Section: Discussionmentioning

confidence: 99%

Yeast inorganic pyrophosphatase. A model for active-site structure based on cadmium-113 and phosphorus-31 NMR studies

Welsh¹,

Cooperman²

1984

Biochemistry

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Equilibrium dialysis and 113Cd2+ NMR studies in the presence of inorganic phosphate (Pi) provide clear evidence for the existence of four well-defined Cd2+ sites per yeast inorganic pyrophosphatase subunit. Parallel 31P NMR studies demonstrate the existence of two binding sites per subunit for Pi and provide strong confirmatory evidence for a small amount of enzyme-bound inorganic pyrophosphate in equilibrium with enzyme-bound Pi. Such inorganic pyrophosphate formation was demonstrated by chemical analysis earlier [Welsh, K.M., Armitage, I.M., & Cooperman, B.S. (1983) Biochemistry 22, 1046-1054]. In this same earlier paper, we provided evidence for inner-sphere contact between enzyme-bound Cd2+ and Pi bound in the higher affinity of the two Pi sites. We now present heteronuclear decoupling evidence that one to three different Cd2+ ions make such contact. The divalent metal ion cofactor conferring the highest activity on inorganic pyrophosphatase is Mg2+, and we present evidence from competition experiments that such inner-sphere contact is also likely for the Mg2+-enzyme. On the other hand, these experiments also show that some metal ion binding sites on the enzyme bind Mg2+ and not Cd2+, and some bind Cd2+ and not Mg2+. These results are considered along with others obtained recently in proposing an active-site structure for inorganic pyrophosphatase.

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Section: Discussionmentioning

confidence: 99%

Yeast inorganic pyrophosphatase. A model for active-site structure based on cadmium-113 and phosphorus-31 NMR studies

Welsh¹,

Cooperman²

1984

Biochemistry

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“…The primary sequence has been determined (Cohen et al, 1978) and an X-ray crystallographic determination of its structure is well underway (Bunick et al, 1978). In addition, important information has been obtained on the binding of divalent metal ions and of pyrophosphate and pyrophosphate analogues port et al, 1973;Baykov & Avaeva, 1974), on the identity of essential amino acids at the active site (Heitmann et al, 1972;Cooperman & Chiu, 1973b;Rapoport et al, 1973;Heitmann & Uhlig, 1974), and on the kinetic (Moe & Butler, 1972a,b;Rapoport et al, 1972) and chemical (Cooperman et al, 1973b;Sperow et al, 1973; Konsowitz & Cooperman, 1976;Avaeva et al, 1977) mechanisms of action.…”

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confidence: 99%

Nuclear magnetic resonance studies of inorganic phosphate binding to yeast inorganic pyrophosphatase

Hamm¹,

Cooperman²

1978

Biochemistry

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Yeast inorganic pyrophosphatase is a dimer of identical subunits. Previous work (Rapoport, T.A., et al. (1973) Eur. J. Biochem. 33, 341) indicated the presence of two different Mn2+ binding sites per subunit. In the present work, the binding of inorganic phosphate to the Mn2+-inorganic pyrophosphatase complex has been studied by 1H and 31P nuclear magnetic resonance. Two distinct phosphate sites have been found, having dissociation constants of 0.24 mM and 18 mM. The Mn2+-31P distance from tightly bound Mn2+ to phosphate bound in the low affinity site (6.2 A) is consistent with outer sphere binding. Binding to both phosphate sites can be simultaneously inhibited by the pyrophosphate analogue, hydroxymethanebisphosphonate, providing evidence for the physical proximity of these two sites. The weaker Mn2+ site is apparently far from both phosphate sites. From the magnitudes of the dissociation constants found for both phosphate and analogue binding and the recent work of P.D. Boyer and his co-workers (private communication) on enzyme-catalyzed phosphate-water exchange, it appears unlikely that the hydrolysis of enzyme-bound pyrophosphate is the rate-determining step in the overall enzymatic catalysis of pyrophosphate hydrolysis, at least when Mn2+ is the required divalent metal ion cofactor.

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On the state of aromatic amino acids in inorganic pyrophosphatase from bakers' yeast

Cited by 2 publications

References 0 publications

Yeast inorganic pyrophosphatase. A model for active-site structure based on cadmium-113 and phosphorus-31 NMR studies

Yeast inorganic pyrophosphatase. A model for active-site structure based on cadmium-113 and phosphorus-31 NMR studies

Nuclear magnetic resonance studies of inorganic phosphate binding to yeast inorganic pyrophosphatase

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