Hydrolysis of Adenosine Triphosphate by Crystalline Yeast Pyrophosphatase

Abstract

Schlesinger and Coon's report that crystalline yeast inorganic pyrophosphatase, in addition to its known ability to hydrolyze inorganic pyrophosphate in the presence of Mg ions, is also able to catalyze the hydrolysis of ATP and ADP in the presence of Zn ions was confirmed. A systematic study showed that the ratio of 370 of PPase-,Mg over ATPase-Zn activities per milligram protein in various preparations of pyrophosphatase obtained in the course of isolation of crystalline pyrophosphatase from baker's yeast was nearly identical in all the preparations, independent of their purity. The course of hydrolysis of ATP by crystalline pyrophosphatase in the presence of Zn was carried out with the aid of ion exchange on Dowex 1. The finding of Schtesinger and Coon that the hydrolysis proceeds from ATP to ADP and then slowly to AMP was confirmed. The kinetics of the first phase of the reaction was found to depend on the molar ratio of Zn/ATP in the reaction mixture. Mg ions in the presence of Zn ions have an accelerating effect on the rate of hydrolysis of ATP. This suggests strongly that both activities--ATPase and PPase--are manifestations of the same active group in the protein molecule of crystalline pyrophosphatase.Crystalline yeast pyrophosphatase isolated in 1952 (1) was found to act as a powerful catalyst for the hydrolysis of sodium or potassium pyrophosphate into orthophosphate ions in the presence of magnesium ions, and also to a lesser degree in the presence of other metallic ions such as M n ++ or Co ++. O n the other hand, organic pyrophosphates such as adenosine triphosphate (ATP) or adenosine diphosphate (ADP) were not affected by comparative concentrations of crystalline pyrophosphatase, either in the presence or in the absence of Mg ++ ions.In a recent publication, however, Schlesinger and Coon (2) reported that A T P and several other organic pyrophosphates were hydrolyzed by relatively high concentrations of crystalline pyrophosphatase provided magnesium ions 3:

Mechanism by which metal cofactors control substrate specificity in pyrophosphatase

“…In agreement with previous reports [7,9,10], k cat values were comparable for the three substrates in the presence of Mn# + , Zn# + and Co# + as cofactors. With Mg# + , k cat increased markedly for Substrate specificity of pyrophosphatase (Figure 2).…”

“…Therefore the inability of the respective physiological cofactors to support the ATPase activity of these enzymes has a clear physiological significance, since it prevents futile ATP hydrolysis. In contrast, non-physiological cofactors, such as Mn# + , Zn# + and Co# + , support both PPase [21] and ATPase activities [9,10] in family I enzymes. The results of the present study, together with those of the structural studies on Y-PPase, provide an explanation for this interesting phenomenon.…”

“…However, no such structures are currently available for Mg-PPase of either family. Interestingly, although Mg# + alone is ineffective as a cofactor in ATP hydrolysis, this metal ion significantly stimulates the Mn# + -supported reaction catalysed by yeast PPase [10]. This implies that only some of the four metal ions bound within the enzyme-substrate complex (Figure 4) are critical in determining substrate specificity.…”

“…However, this specificity is lost when transition metal ions such as Zn# + , Mn# + and Co# + are used as cofactors. Family I PPase displays high catalytic activity against PP i in the presence of these metal ions, markedly increased activity against polyphosphates [7] and the ability to hydrolyse organic tri-and diphosphates, such as ATP and ADP [9,10]. ATP hydrolysis by PPase yields ADP as the primary product, which is slowly converted into AMP [7].…”

Inorganic pyrophosphate in plasma in normal persons and in patients with hypophosphatasia, osteogenesis imperfecta, and other disorders of bone

“…Yeast inorganic pyrophosphatase (12) was used to check the specificity of the method. This enzyme is highly specific for PP,, but under special conditions it will hydrolyze ATP (13 When hydrolysis of the 'P [PP,] in the plasma extract was practically complete, the solution was applied immediately to the ion-exchange columns, and the residual phosphate and radioactivity measured in fractions eluting with 0.25 N HCl between 100 and 117 ml (Fig. 1).…”