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Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker's yeast.

ABSTRACT: 1. Pyruvate carboxylase from baker's yeast acts with either MgATP(2-) or MnATP(2-) as substrate. The optimum pH for the enzyme reaction is 8.0 with MgATP(2-) and 7.0 with MnATP(2-). 2. When the reaction velocity is plotted against MgATP(2-) (or MnATP(2-)) concentration slightly sigmoid curves are obtained, either in the presence or in the absence of acetyl-CoA (an allosteric activator). In the presence of excess of free Mg(2+) (or Mn(2+)) the curves turn into hyperbolae, whereas in the presence of excess of free ATP(4-) the apparent sigmoidicity of the curves increases. 3. The sigmoidicity of the plots of v against MgATP(2-) (or MnATP(2-)) concentration can be explained by the inhibitory effect of free ATP(4-), the concentration of which, in the experimental conditions employed, is significant and varies according to the total concentration of the ATP-magnesium chloride (or ATP-manganese chloride) mixture. Free ATP(4-) behaves as a negative modifier of yeast pyruvate carboxylase. 4. The effect of high concentrations of Mg(2+) (or Mn(2+)) on the kinetics of yeast pyruvate carboxylase can be explained as a deinhibition with respect to ATP(4-), instead of a direct enzyme activation. 5. At pH6.5 manganese chloride is more effective than magnesium chloride as enzyme activator even in the presence of a great excess (16-fold) of the latter. This is consistent with a significant contribution of the MnATP(2-) complex to the activity of yeast pyruvate carboxylase, in medium conditions resembling those existing inside the yeast cell (pH6.25-6.75; 12mm-magnesium chloride and 0.75mm-manganese chloride). 6. The physiological significance of the enzyme inhibition by free ATP(4-) is doubtful since the Mg(2+) and Mn(2+) concentrations reported to exist inside the yeast cell are sufficient to decrease ATP(4-) concentrations to ineffective values.

SUBMITTER: Cazzulo JJ

PROVIDER: S-EPMC1187779 | biostudies-other | 1969 May

REPOSITORIES: biostudies-other

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Project description:1. The effects of ATP, PP(i) and EDTA on the skeletal-muscle pyruvate kinase reaction at various concentrations of magnesium (where ;magnesium' refers to total Mg(2+), both free and in the form of complexes) were investigated. The reaction rate was determined as the amount of pyruvate formed in a recorded time of incubation. 2. At 44mm-magnesium the K(m) values for ADP and phosphoenolpyruvate were unaltered by the presence of ATP up to 6.8mm in systems buffered with either tris-hydrochloric acid or glycylglycine-sodium hydroxide, but the K(m) values were different in these systems. The K(m) for one substrate was independent of the concentration of the second substrate. 3. At 10mm-magnesium in the tris-hydrochloric acid system ATP inhibited the reaction competitively with respect to ADP and phosphoenolpyruvate. In the glycylglycine-sodium hydroxide system the inhibition appeared to be non-competitive. At 10mm-magnesium the K(m) values were lower than at 44mm-magnesium and dependent on the system used. 4. In the tris-hydrochloric acid system the reaction rate rose with increasing magnesium concentration up to a maximum at a concentration 10-20 times that of ADP. Further increase inhibited the reaction and at 44mm-magnesium the rate was 25-50% of its maximum. This inhibition paralleled that produced by increasing trimethylammonium chloride concentrations and was not due to a specific effect of the Mg(2+) ion. 5. In the presence of 6.8mm-ATP no reaction occurred below 4-6mm-magnesium, and further increase apparently abolished the inhibition as the reaction rate increased and became equal to those obtained in the absence of ATP at 10-25mm-magnesium. Further increase in magnesium concentration gave reaction rates that were slightly higher in the presence of ATP than in its absence. The maximal rate in the presence of ATP was distinctly lower than in its absence. When 6.8mm-PP(i) or 6.8mm-EDTA was present the variations in reaction rate with rising magnesium concentration were similar to that obtained in the presence of ATP below 6-8mm-magnesium but further increase in the magnesium concentration resulted in an increase in the rate up to a maximum comparable with that of the control. The effect of pure chelation was thus a displacement of the reaction maximum to higher magnesium concentrations without changing the maximal rate. When correction had been made for this effect, ATP gave inhibition at 44mm-magnesium that was competitive with respect to ADP (K(i) 2.1x10(-2)m). This degree of inhibition is far less than was reported earlier and its importance for the mechanism of the pyruvate kinase reaction is discussed.

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Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker's yeast.

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