Phosphofructokinase type 1 kinetics, isoform expression, and gene polymorphisms in cancer cells.
Kinetic analysis of PFK-1 from rodent AS-30D, and human HeLa and MCF-7 carcinomas revealed sigmoidal [fructose 6-phosphate, Fru6P]-rate curves with different V(m) values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH(4)(+), or K(+). The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non-essential hyperbolic activation. PFK-1 from rat liver and heart also exhibited the mixed cooperative-hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased V(m) and increased content of inactive (T) enzyme forms. High K(+) prompted increased (Fru6P) or decreased (activators) affinities; increased V(m); and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK-1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF-7 cells, of L>M in AS-30D cells, and of C in HeLa cells. PFK-1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK-1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK-1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK-1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux-control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect.
Departamento de Bioquímica, Instituto Nacional de Cardiología, México DF 14080, Mexico. rafael.moreno@cardiologia.org.mx
Rafael
R
Moreno-Sánchez
Alvaro
A
Marín-Hernández
Juan Carlos
JC
Gallardo-Pérez
Héctor
H
Quezada
Rusely
R
Encalada
Sara
S
Rodríguez-Enríquez
Emma
E
Saavedra
D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms / Q000201:enzymology / Q000235:genetics; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008114:Liver Neoplasms, Experimental / Q000201:enzymology / Q000235:genetics; D009206:Myocardium / Q000201:enzymology; D009369:Neoplasms / Q000201:enzymology* / Q000235:genetics*; D010732:Phosphofructokinase-1 / Q000235:genetics / Q000378:metabolism*; D025501:Phosphofructokinase-1, Liver Type / Q000235:genetics / Q000378:metabolism; D025203:Phosphofructokinase-1, Muscle Type / Q000235:genetics / Q000378:metabolism; D025502:Phosphofructokinase-1, Type C / Q000235:genetics / Q000378:metabolism; D011110:Polymorphism, Genetic; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D002583:Uterine Cervical Neoplasms / Q000201:enzymology / Q000235:genetics
D018076:DNA, Complementary; D025501:Phosphofructokinase-1, Liver Type; D025203:Phosphofructokinase-1, Muscle Type; D025502:Phosphofructokinase-1, Type C; D010732:Phosphofructokinase-1