Activation of Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase by inorganic phosphate.
Pseudomonas ochraceae 4-hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17], one of the metal ion-requiring aldolases, is markedly activated by Pi. The activation is reversible and can be observed in every step of enzyme purification. The extent of activation is almost independent of the metal ion used, but varies with each substrate. The cleavage of l-4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, is most strongly activated: Pi gives a hyperbolic activation curve with an activation constant of 0.36 mM and a maximum activation of about 65-fold. Arsenate, phosphorous acid, bicarbonate, acetyl phosphate, thiamine diphosphate, ADP, PPi, and ATP are also effective to various extents. These anions appear to be effective in the free form but not in the metal ion-complex. Many organic and inorganic anions are ineffective. Pi causes parallel increases in Vmax and in Km for substrate or metal ion with a concomitant shift of the optimum pH toward the alkaline side, and the enhancement of activity is closely correlated with the shift of optimum pH. Pi induces no gross change of molecular form of the enzyme protein as evaluated from gel filtration, PAGE, UV, fluorescence, and CD spectral data. Based on these findings, the mechanism and the physiological meaning of the observed activation are discussed.
Maruyama
K
K
Department of Chemistry, Faculty of General Education, Gifu University.
D002850:Chromatography, Gel; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D008956:Models, Chemical; D008970:Molecular Weight; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology*; D011549:Pseudomonas / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D008670:Metals; D010710:Phosphates; D007652:Oxo-Acid-Lyases; C021412:4-hydroxy-4-methyl-2-oxoglutarate aldolase; D008274:Magnesium