Kinetic properties of pyruvate kinase in human maternal leukocytes in fetal malnutrition

Pyruvate kinase (PK) is one of the regulatory enzymes in glycolysis. The present study was undertaken to determine whether regulation of the enzyme by normally occurring metabolites was disturbed in leukocytes of mothers who delivered fetally malnourished (FM) babies. Kinetic studies of enzyme regulation by physiologic effectors approximated a potential regulating mechanism of the enzyme in its cellular environment. There are two isoenzymes of PK. Leukocytes contain an M2 enzyme with intermediate regulatory properties between the liver (type L) and the muscle (type M) enzymes. The presence in the cell of M2 PK in the A form leads to inhibition of glycolysis by amino acids, such as alanine, and therefore, to the sparing of glucose but probably inhibition of energy production from glucose. In this study, leukocytes were isolated from blood of six pregnant women and 11 women in the postpartum period in Oklahoma and at parturition from 31 women in Mexico. Fourteen of the latter group delivered FM babies. The kinetic characteristics of the nonpurified enzyme PK with respect to allosteric modulation in fructose-1, 6-phosphate (FDP) and L-alanine (Ala) were studied in the leukocyte extracts. Data for initial reaction velocities (v) vs substrate concentrations (s), double reciprocal Lineweaver-Burk plots, and Hill plots are presented. The equations for the double reciprocal plots were determined by linear regression analysis. The enzyme constants were derived by computer, and the values compared by the Mann-Whitney U-test. In all subjects studied, 0.5 mM FDP activated and 2 mM L-alanine inhibited the enzyme. During pregnancy, the v vs s concentration curves were hyperbolic (Hill coefficient, n is less than 1.0) except for the Ala-inhibited enzyme during pregnancy, which had a sigmoid curve, n=1.54. The interaction of FDP and Ala was dependent on the concentration of the substrate phosphoenolpyruvate (PEP) at low [PEP]. There was net activation, not inhibition, at high concentrations; the switchover was at 0.5 mM PEP during pregnancy. In Mexican mothers having normal babies (normal mothers) the maximum initial velocity, V (micromoles per min per mg of DNA), with respect to PEP, was 2.22+/-0.34; in FM mothers, V was 2.01+/-0.44. With respect to binding of the substrate, PEP, V of the leukocyte enzymes in FM mothers vs normal mothers was equally inhibited by Ala (deltaV=-50% vs -47%), but was significantly less responsive to stimulation by FDP (deltaV=+10% vs +75%). When both Ala and FDP were present, FDP less effectively overcame the inhibition by Ala (deltaV=-9% vs +54%). The K0.05 of the enzyme (molar concentration X 10(-4) PEP) was significantly reduced by FDP, whether Ala was present or not, during pregnancy and in the postpartum period in leukocytes of Oklahoma mothers and at term in Mexican mothers. The K0.5 for normal and FM mothers was similar. Thus, the enzyme in leukocytes of Mexican mothers who delivered FM and normal babies exhibited different kinetic responses to the allosteric modulators...