NUCLEOTIDE SPECIFICITY OF OXALACETIC CARBOXYLASE

The reversible oxidation of 3α-hydroxysteroids to their corresponding 3-keto forms comprises an important step in the metabolism of C19-steroids. The described techniquue demonstrates the activity of the enzyme catalyzing this reaction, with the use of androsterone as a substrate and a tetrazolium salt as the final electron acceptor. The enzyme is specific for 3α-hydroxysteroids; there was no histochemical reaction with epiandrosterone, the β isomer of androsterone. Since 3α-hydroxysteroid dehydrogenase is soluble in aqueous solutions, it was necessary to increase the osmolarity of the incubation medium by adding polyvinylpyrrolidone in a final concentration of 20%. Although the enzyme has a dual nucleotide specificity, no appreciable differences were seen in its distribution pattern in rat tissues with either NAD or NADP as a coenzyme. In adult female rats, enzyme activity was present in the liver, kidneys and clitoral glands. In mature males, diformazan deposits were observed in the liver, kidneys, preputiai glands, epididymis, ventral prostate and Leydig cells.

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Kinetic Studies of Yeast PolyA Polymerase Indicate an Induced Fit Mechanism for Nucleotide Specificity†

Biochemistry ◽

10.1021/bi050089r ◽

2005 ◽

Vol 44 (21) ◽

pp. 7777-7786 ◽

Cited By ~ 14

Author(s):

Paul B. Balbo ◽

Gretchen Meinke ◽

Andrew Bohm

Keyword(s):

Kinetic Studies ◽

Induced Fit ◽

Nucleotide Specificity

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Pyruvatephosphate dikinase from Bacteroides symbiosus

Biochemical Journal ◽

10.1042/bj1250531 ◽

1971 ◽

Vol 125 (2) ◽

pp. 531-539 ◽

Cited By ~ 22

Author(s):

Richard E. Reeves

Keyword(s):

Metal Ion ◽

Adenine Nucleotides ◽

Thiol Compounds ◽

Pyruvate Phosphate Dikinase ◽

Constant Ph ◽

Bacterial Enzyme ◽

Rate Of Reaction ◽

Ph Stat ◽

Magnesium Salts ◽

Nucleotide Specificity

1. An improved method is given for preparation of pyruvate, phosphate dikinase from Bacteroides symbiosus. 2. The bacterial enzyme is stable, free from interfering enzyme activities, and does not require thiol compounds to maintain stability during storage or assay. 3. New direct assays of enzyme activity are based on acid evolution or consumption as measured at constant pH in a pH-stat. 4. The optimum rate of reaction in the direction of pyruvate formation occurs at about pH6.4; in the direction of phosphoenolpyruvate formation, it is at pH7.2–7.8. 5. Newly determined substrate Km values for the enzyme are: AMP, 3.5×10−6m; ATP, 1×10−4m; pyruvate, 8×10−5m; Pi, 6×10−4m. 6. K+ may substitute for NH4+ in activating the reaction catalysed by the B. symbiosus enzyme. 7. In the direction of pyruvate formation the bivalent metal ion requirement of the enzyme is fulfilled by salts of nickel, manganese, magnesium and cobalt. In the other direction only magnesium salts were effective. 8. The nucleotide specificity of the enzyme is strictly limited to the adenine nucleotides. CTP and ITP strongly inhibit the reaction in the direction of phosphoenolpyruvate formation.

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