5-Oxoprolinase from rat kidney, I. Assay, purification, and determination of kinetic parameters.
A new assay for the determination of 5-oxoprolinase activity is described. The enzyme 5-oxoprolinase was purified from rat kidney 285-fold to apparent homogeneity, as judged by analytical disc electrophoresis and discontinous polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The specific activity of the preparation was 122 mU/mg of protein. A complete initial rate kinetic analysis of the forward reaction catalyzed by 5-oxoprolinase was carried out using 5-oxo-L-proline and MgATP2theta as substrates. The computer-fitted double reciprocal plots showed intersecting patterns indicating a sequential mechanism. The data were fitted by weighted linear regression analysis using the complete equation for bisubstrate reactions. The limiting Michaelis constants for 5-oxoproline and MgATP2theta were calculated to be 31.6 +/- 2.3 muM and 172.7 +/- 11.5muM, respectively. The maximum forward rate is 1.2 +/- 0.02 mumol X min-1; the turnover number 7.0 min-1.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008433:Mathematics; D011761:Pyrrolidonecarboxylic Acid; D051381:Rats
D000255:Adenosine Triphosphate; D000581:Amidohydrolases; D008274:Magnesium; D011761:Pyrrolidonecarboxylic Acid