A comparative study of phosphofructokinase and tagatose 6-phosphate kinase from streptococcus lactis : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University
In the lactic streptococci glucose is metabolised to lactic acid via the Embden-Meyerhof-Parnas (EMP) pathway. Metabolism of lactose and galactose in these organisms involves participation of the D-tagatose 6-phosphate pathway in which galactose 6-phosphate is metabolised to triose phosphates via tagatose derivatives. Phosphofructokinase (ATP : D-fructose 6-phosphate 1-phosphotransferase, E.C. 184.108.40.206) catalyses the ATP-dependent phosphorylation of fructose 6-phosphate in the EMP pathway. The analogous reaction in the tagatose 6-phosphate pathway, phosphorylation of tagatose 6-phosphate with ATP, is catalysed by a specific enzyme, tagatose 6-phosphate kinase. While phosphofructokinase (PFK) is known to be a major regulatory enzyme in carbohydrate metabolism in most organisms, little is known of the regulatory properties of tagatose 6-phosphate kinase (T6PK). PFK and T6PK were purified from Streptococcus lactis C10 PFK was purified to homogeneity (364-fold purification) by affinity chromatography on Blue-dextran-Sepharose. Unlike PFK, T6PK did not bind to Blue-dextran-Sepharose : a 136-fold purification was achieved using ammonium sulphate fractionation, gel filtration, and ion exchange chromatography. A study of some of the properties of PFK and T6PK from S. lactis C10 showed that these two enzymes are distinct proteins with different physical and kinetic characteristics. S. lactis PFK is a tetramer (MW 145,000 daltons) of identical subunits of molecular weight 33,500 daltons. It therefore appears structurally similar to other bacterial PFKs. T6PK from S. lactis has a molecular weight of approximately 114,000 daltons, a value similar to that of Staphylococcus aureus T6PK which is a dimer. S. lactis PFK exhibited the co-operative binding of F6P and inhibition by high concentrations of ATP relative to F6P which is typical of most bacterial and mammalian PFKs. F6P0.5. and Km (MgATP) values were 0.28 mM and 0.18 mM respectively. ADP stimulated PFK activity, shifting the sigmoidal saturation curve to a more hyperbolic form, with a corresponding decrease in nH. Ammonium and potassium ions also activated PFK, while activity was inhibited by AMP, PEP, FBP, T6P and inorganic phosphate. In contrast to PFK, T6PK showed no co-operative binding of sugar phosphate substrate and was less sensitive than PFK to ATP inhibition. Km values for T6P and MgATP were 0.16 mM and 0.4 mM respectively. Apart from ammonium and potassium ions, no activators of T6PK were found. Activity was inhibited by ADP, PEP, and FBP. PFK and T6PK could catalyse phosphorylation of both F6P and T6P although the enzymes showed a much greater affinity for their natural substrate. Maximum velocities attained were higher with the natural substrate than when the other sugar phosphate was used as substrate. Both enzymes showed similar pH optima and divalent cation requirement. Levels of PFK, T6PK, and Galactokinase (Gal K), enzymes of the Embden-Meyerhof-Parnas, Tagatose 6-phosphate, and Leloir pathways respectively, were measured in strains of S. lactis, S. cremoris, S. diacetylactis and S. faecalis grown on different sugars. Growth on lactose and galactose induced increased levels of T6PK and Gal K activity, galactose generally inducing higher levels of T6PK than lactose. In most strains, addition of glucose to media containing lactose or galactose resulted in lowered activities of Gal K, comparable to those in glucose-grown cells. In contrast, T6PK activity was generally not suppressed by growth on glucose plus lactose, while in growth on glucose plus galactose, T6PK activity was approximately 50% of the activity in cells grown on glucose alone. PFK activity was generally unaffected by the sugar in the growth medium. In spite of changes in specific activities of PFK and T6PK throughout the growth period of S. lactis, the ratio of PFK : T6PK remained fairly constant. The properties of S. lactis PFK and T6PK are compared to those of these enzymes in other bacteria, and the possible role of T6PK in regulation of carbohydrate metabolism in S. lactis is discussed.