KINETICS OF OVEREXPRESSED TRANSKETOLASE FROM ESCHERICHIA-COLI JM-107 PQR-700/

Initial rate and product inhibition studies using pH-stat unbuffered b iotransformation mixtures at 25 degrees C and pH 7.0 have been perform ed on overexpressed transketolase (EC 2.2.1.1.) from a self-cloned tra nsformant of Escherichia coli. The Michaelis and inhibition constants for the cosubstrates, hydroxypyruvate and glycolaldehyde, and the inhi bition constant of the L-erythrulose product were determined. The kine tic data are consistent with a Ping Pong Bi Bi mechanism where the ket ol donor (hydroxypyruvate) is first bound to the enzyme and is followe d by release of the first product CO,, followed by the binding of the aldehyde acceptor (glycolaldehyde) and subsequent release of the secon d product, L-erythrulose. The enzyme was shown to be free from excess substrate inhibition up to 40 mmol l(-1) for hydroxypyruvate and 100 m mol l(-1) for glycolaldehyde. In addition, inhibition by the L-erythru lose product in conformity with the predicted mechanism was found to b e competitive. (C) 1997 by Elsevier Science Inc.