No abstract

Using purified enzyme and homologous tRNA, we have investigated the order of substrate binding and product release for the human placental threonyl-tRNA synthetase by isotope exchange, initial velocity, dead-end inhibition and product inhibition studies. The kinetic patterns obtained from these studies are consistent with a unique Bi Uni Uni Bi ping-pong mechanism. The order of addition of the first two substrates ATP and threonine is random, while the release of products follows an obligatory sequence, with AMP as the last product to dissociate from the enzyme.

Human threonyl-tRNA synthetase has been purified from full-term placenta over 5000-fold with a 13% overall yield by a combination of affinity chromatographic methods and conventional procedures. The product was apparently homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the esterification of -3500nmol L-threonine t o tRNA per min per mg of protein at 37O, corresponding t o a molecular activity of -700/min. The apparent molecular weight of the native enzyme ranged from 210 000 to 220 000 by gel-filtration on Sephadex G-200, and was either -11 0 000 or 228 000 by sucrose gradient centrifugation for different preparations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated a single band of molecular weight 85 000 or 1 15 000 for different preparations. These results suggest that human placental threonyl-tRNA synthetase has a subunit structure of the type a2 with a subunit molecular weight of about 100000. However, some other molecular forms with lower specific activity were also found t o exist under certain conditions.