Pig liver phosphomevalonate kinase. 2. Participation of cysteinyl and lysyl groups in catalysis

Sergio Bazaes, Enrique Beytía, Ana María Jabalquinto, Francisco Solís De Ovando, Isabel Gómez, Jaime Eyzaguirre

Resultado de la investigación: Contribución a una revistaArtículorevisión exhaustiva

12 Citas (Scopus)

Resumen

Phosphomevalonate kinase from pig liver is inactivated by 5,5′-dithiobis(2-nitrobenzoate) and pyridoxal 5′-phosphate. The substrate phosphomevalonate protects the enzyme against inactiyation by these reagents. Inactivation by 5,5′-dithiobis(2-nitrobenzoate) is complete and may be reverted by 2-mercaptoethanol or dithiothreitol. Experiments carried out with partially inactivated enzyme show no change in the kcat or in the apparent Km for the substrates, as compared with the native enzyme, indicating the existence of two populations of molecules, one intact and the other totally inactive. These results suggest that 5,5′-dithiobis(2-nitrobenzbate) reacts with the only cysteinyl residue of the enzyme and that this residue is located in or near the active site. Inhibition by pyridoxal 5′-phosphate can be reverted, either by dialysis or by the addition of lysine, but not if the partially inactivated enzyme is treated previously with NaBH4, in agreement with the formation of a Schiff base between pyridoxal 5′-phosphate and an amino group of the enzyme. This is further supported by the appearance of an absorption band with a maximum at 325 nm in the enzyme treated with pyridoxal 5′-phosphate and NaBH4. Pyridoxal and pyridoxamine 5′-phosphate are weaker inhibitors than pyridoxal 5′-phosphate, suggesting a specific effect due to the phosphate and aldehyde groups. The enzyme is not completely inactivated by pyridoxal 5′-phosphate, even at a molar ratio of 350, or by a second inactivation treatment after reduction with NaBH4. The partially modified enzyme shows a lower Km for phosphomevalonate than the native enzyme, suggesting that the reactive group is located near the binding site of phosphomevalonate. The lower Km may reflect an effect of the positive charge of the pyridoxal 5′-phosphate ring nitrogen, enhancing the binding of phosphomevalonate. Values of 8.15 at 24°C and 7.95 at 31°C have been determined for the pK of the reactive group. A ΔHi of 11.8 kcal/mol has been estimated, in agreement with the values expected for an amino group. One amino group per active site is involved in the enzyme inactivation as shown by kinetic data. Quantification of the number of moles of pyridoxal 5′-phosphate bound per mole of enzyme is not conclusive but supports this assertion. This group may correspond to an ∈-amino group of lysine.

Idioma originalInglés
Páginas (desde-hasta)2305-2310
Número de páginas6
PublicaciónBiochemistry
Volumen19
N.º11
EstadoPublicada - 1980

Áreas temáticas de ASJC Scopus

  • Bioquímica

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