Human pyruvate kinases. Role of the divalent cation in the catalytic mechanism of the red cell enzyme

Abstract

The kinetic properties of human erythrocyte pyruvate kinase vary depending on whether Mg2+ or Mn2+ is used as the essential divalent cation. In the Mg2+-activated system catalytic activity is dependent on MgADP but not on the concentrations of uncomplexed Mg2+ or ADP. The Mn2+activated reaction is dependent on both free Mn2+ and Mn-ADP, but not on uncomplexed ADP. Free P-enolpyruvate, rather than P-enolpyruvate complexed with metal, appears to be the preferred substrate. The velocity profile for P-enolpyruvate in the Mg2+-activated system is sigmoid (Hill's = 1.78) and the addition of low concentrations of fructose-1,6-P shifts the profile to a hyperbola ( = 0.92). In the Mn2+activated reaction a single hyperbolic curve is obtained with or without fructose-1,6-P ( = 0.95). Hill plots for free Mg2+ and free Mn2+ show slopes of 1.2 and 1.7, respectively. At Xyruvate kinase (EC 2.7.1.40) catalyzes the conversion of P-enolpyruvate1 and ADP to pyruvate and ATP; the reaction requires both monovalent and divalent cations (Boyer et al., 1943). Cationic cofactors are required by many enzymes and apparently play a significant role in metabolic regulation (Wyatt, 1964;Bygrave, 1967). The observations that the cation specificity of glutamine synthetase changes from Mg2+ to Mn2+ on adenylylation (Kingdon et al., 1967;Shapiro and Ginsburg, 1968), that Escherichia coli ADP: glucose synthetase has markedly different kinetics depending on whether Mg2+ or Mn2+ serves as cofactor (Centner and Preiss, 1968) and that yeast phosphofructokinase shows a dependence on both uncomplexed and complexed Mg- (Mavis and Stellwagen, 1970) indicate the importance of specific cation effects.A recent estimate of intracellular concentrations of free Mg2+ (Rose, 1968) in the human erythrocyte suggests that substantial amounts of ATP and ADP exist in the uncomplexed form, emphasizing the need to examine the effects of both free and bound ligands on red cell enzyme catalysis. The present communication describes the kinetic differences between Mg2+ and Mn2+ activation of a pyruvate kinase isolated from human erythrocytes.Experimental Section Methods Materials. DPNH, Na2ADP, trisodium P-enolpyruvate, and crystalline rabbit muscle pyruvate kinase, lactate det From the

Thermodynamic Linked-Function Analysis of Mg2+-Activated Yeast Pyruvate Kinase

“…Studies with the human erythrocyte (type R) pyruvate kinase indicate that Mn 2+ plays a role similar to that of FBP. A single hyperbolic curve is obtained in the presence and absence of FBP with this enzyme in the presence of Mn 2+ (13).…”

The influence of divalent cations on allosteric behaviour of muscle pyruvate kinase from the sea mussel Mytilus edulis L.

“…Recently it has been shown (Leonard, 1972) that for erythrocyte pyruvate kinase Mn 2÷ changes the sigmoidal substrate saturation curve into a hyperbole and causes desensitization for FDP. The results presented indicate that changes of pyruvate kinase activity may be the result not only of variations in the concentration of organic substances such as PYR-P, ATP, alanine and FDP, but also in the intracellular concentrations of metal ions.…”

Regulatory Properties of Three Human Pyruvate Kinases

“…Erythrocyte PK I was prepared from outdated Red Cross blood as described by Leonard [17]. Briefly, the procedure involved washing red cells, hemolysis in distilled water, adsorption onto and batchwise elution from DEAE cellulose, precipitation with (NH4)2S04 between 20 and 38% saturation, heating at 40°C for 1 h in pH 5.5 citratephosphate buffer containing 1 mmol/1 K2EDTA and 1 mmol/1 mercaptoethanol, and a second precipitation with (NH4)2S04 between 20 and 38% saturation.…”