Phosphorylation is switch of L-type pyruvate kinase allostery

Among four pyruvate kinase isoenzymes, M1, M2, R and L, only M1 is considered as a nonallosteric enzyme. However, here we show that the non-phosphorylated L-type pyruvate kinase (L-PK) is also a non-allosteric enzyme with respect to its substrate phosphoenolpyruvate (PEP). The allosteric catalytic properties of L-PK are switched on through phosphorylation by cAMP-dependent protein kinase. The non-phosphorylated enzyme was produced by expressing the rat L-PK in E. coli, as the bacterium does not have mammalian-type protein kinases. The resulting tetrameric protein was phosphorylated with a stoichiometric ratio of one mole of phosphate per one L-PK monomer. Activity of the phosphorylated enzyme was allosterically regulated by PEP with the Hill coefficient n=2.5. It was observed that allostery was engaged by phosphorylation of the first subunit in the tetrameric enzyme, while further phosphorylation only modulated this effect. The discovered switching between non-allosteric and allosteric forms of L-PK and the possibility of modulating the allostery by phosphorylation are important for understanding of the interrelationship between allostery and the regulatory phosphorylation in general, and may have implication for further analysis of glycolysis regulation in the liver.

Participation of the Entner–Doudoroff pathway inEscherichia colistrains with an inactive phosphotransferase system (PTS–Glc+) in gluconate and glucose batch cultures

The activity of the enzymes of the central metabolic pathways has been the subject of intensive analysis; however, the Entner–Doudoroff (ED) pathway has only recently begun to attract attention. The metabolic response to edd gene knockout in Escherichia coli JM101 and PTS–Glc+was investigated in gluconate and glucose batch cultures and compared with other pyruvate kinase and PTS mutants previously constructed. Even though the specific growth rates between the strain carrying the edd gene knockout and its parent JM101 and PTS–Glc+edd and its parent PTS–Glc+were very similar, reproducible changes in the specific consumption rates and biomass yields were obtained when grown on glucose. These results support the participation of the ED pathway not only on gluconate metabolism but on other metabolic and biochemical processes in E. coli. Despite that gluconate is a non-PTS carbohydrate, the PTS–Glc+and derived strains showed important reductions in the specific growth and gluconate consumption rates. Moreover, the overall activity of the ED pathway on gluconate resulted in important increments in PTS–Glc+and PTS-Glc+pykF mutants. Additional results obtained with the pykA pykF mutant indicate the important contribution of the pyruvate kinase enzymes to pyruvate synthesis and energy production in both carbon sources.Key words: Escherichia coli, gluconate metabolism, Entner-Doudoroff pathway, PT system, pyruvate kinase isoenzymes.

PYRUVATE KINASE ISOENZYMES FROM SUBMAXILLARY AND SUBLINGUAL SALIVARY GLANDS OF RAT (Rattus rattus norvaegicus, Berkenhout). I PURIFICATION AND PHYSICOCHEMICAL PROPERTIES

Piruvatoquinase de glândula salivar submaxilar e sublingual de rato (Rattus rattus norvaegicus, Berkenhout) foi purificada até homogeneidade por “salting out” por precipitação com sulfato de amônio seguida de cromatografia de coluna, primeiro com fosfocelulose e eluição com solução 0,5M de KCl e depois com Blue Sepharose CL-6B eluida com PEP 5mM e ADP 5mM. Obteve-se atividade específica final de 324,5 UI/mg com rendimento global de 20,1% para SM-PK e de 427,4 UI com rendimento global de 9,5% para SL-PK, com pesos moleculares de 60.500 e 50.000 Daltons determinado em eletroforese do tipo PAGE com e sem SDS, para SM-PK e de 242.000 e 200.000 para SL-PK, sugerindo, com isso que se tratam de homotetrâmeros. Por eletroforese em gel de acetato demonstrou-se que tanto SM-K como SL-K possuem somente uma forma isoenzimática com mobilidade eletroforética similar à PK tipo L de fígado de rato e do tipo M2 do rim de rato. verificou-se que o pH ótimo para ambas as enzimas é de 7,4. Abstract Pyruvate kinase from rat (Rattus rattus norvaegicus) submaxillary and sublingual salivary glands was purified to homogeneity by a 3-step process. One step involved salting out by ammonium sulfate precipitation and two steps, column chromatography, first with phosphocellulose and elution with 0.5M KCl and then with Blue Sepharose CL-6B eluted with 5mM PEP and 5 mM ADP. The final specific activity of SM-PK was 324.5 IU/mg with an overall yield of 20.1%. The values for SL-PK were 427. 4 IU/mg and a yield of 9.5%. The molecular weights of the native enzymes and their subunits, as determined by PAGE electrophoresis with or without SDS were 60.500 and 50.000 Daltons respectively, for SM-PK and 242.000 and 200.000 for SL-PK, suggesting that these enzymes were present as homotetramers. By means of cellulose acetate electrophoresis it has been demonstrated that both SM-PK and SL-PK possess only one isozymic form displaying eletrophoretic mobility similar to that of the L-type PK from rat liver and M2- type PK form rat kidney. Optimum pH for both SM-PK and SL-PK was found to be 7. 4 in Tris-HCl buffer.