Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae grown on phthalate.
4-Hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] has been purified to homogeneity (about 770-fold purification, yield 11.4%) from Pseudomonas ochraceae grown on phthalate. The enzyme has a molecular weight of 160,000 (gel filtration on Bio-Gel A-1.5m), a subunit molecular weight of 26,000 (SDS-PAGE) and an isoelectric point of 5.0 (isoelectric focusing). The enzyme requires divalent metal ions such as Mg2+, Mn2+, Co2+, Zn2+, and Cd2+ for activity. The enzyme actively cleaves 4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, to give pyruvate and oxaloacetate, but shows much lower affinity for 4-hydroxy-4-methyl-2-oxoglutarate. 4-Hydroxy-2-oxoglutarate is cleaved at a low rate to pyruvate and glyoxylate. The l-isomers of the substrates are preferentially cleaved rather than the d-isomers as determined polarimetrically. The enzyme reactions are reversible: the equilibrium constants (pH 8.0, 25 C) for the HMG and HG cleavage reactions are about 0.07 and 0.03 M, respectively, whereas no equilibrium is observed with CHA due to oxaloacetate beta-decarboxylase activity associated with the enzyme. The enzyme activity is hardly affected by thiols and thiol reagents. The non-enzymatic cleavage reaction caused by various metal ions has also been studied to examine the mechanistic similarity to the enzymatic reaction.
Maruyama
K
K
Department of Chemistry, Faculty of General Education, Gifu University.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D009904:Optical Rotation; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010795:Phthalic Acids; D011549:Pseudomonas / Q000201:enzymology*; D013237:Stereoisomerism
D008670:Metals; D010795:Phthalic Acids; D005634:Fructose-Bisphosphate Aldolase; D007652:Oxo-Acid-Lyases; C021412:4-hydroxy-4-methyl-2-oxoglutarate aldolase