Effects of pressure on enzyme function of Escherichia coli dihydrofolate reductase

Ohmae Eiji

Tatsuta Mineyuki

Abe Fumiyoshi

Kato Chiaki

Tanaka Naoki

Kunugi Shigeru

Gekko Kunihiko

Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics

To elucidate the effects of pressure on the function of Escherichia coli dihydrofolate reductase (DHFR), the enzyme activity and the dissociation constants of substrates and cofactors were measured at pressures up to 250 MPa at 25ºC and pH 7.0. The enzyme activity decreased with increasing pressure, accompanying the activation volume of 7.8 ml mol-1. The values of the Michaelis constant (Km) for dihydrofolate and NADPH were slightly higher at 200 MPa than at atmospheric pressure. The hydride-transfer step was insensitive to pressure, as monitored by the effects of the deuterium isotope of NADPH on the reaction velocity. The dissociation constants of substrates and cofactors increased with pressure, producing volume reductions from 6.5 ml mol-1 (tetrahydrofolate) to 33.5 ml mol-1 (NADPH). However, the changes in Gibbs free energy with dissociation of many ligands showed different pressure dependences below and above 50 MPa, suggesting conformational changes of the enzyme at high pressure. The enzyme function at high pressure is discussed based on the volume levels of the intermediates and the candidates for the rate-limiting process.

Dihydrofolate reductase

Enzyme activity

High pressure

Volume change

Biology [ 460 ]

eng

Elsevier Science BV

Copyright (c) 2008 Elsevier B.V.

[ISSN] 1570-9639

[DOI] 10.1016/j.bbapap.2008.04.005

[NCID] AA11685085

[DOI] http://dx.doi.org/10.1016/j.bbapap.2008.04.005