TY - JOUR
T1 - Nucleotide binding affinites of the intact proton-trans locating transhydrogenase from Escherichia coli
AU - Bizouarn, T
AU - van Boxal, GI
AU - Bhakta, T
AU - Jackson, John
PY - 2005/7/15
Y1 - 2005/7/15
N2 - Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K-d values) for NADPH (0.87 mu M), NADP(+) (16 mu M), NADH (50 mu M), and NAD(+) (100-500 mu M) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K-d values for NAD(+) and NADH are similar to those previously reported with isolated dI, but the K-d values for NADP(+) and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidised. (c) 2005 Elsevier B.V. All rights reserved.
AB - Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K-d values) for NADPH (0.87 mu M), NADP(+) (16 mu M), NADH (50 mu M), and NAD(+) (100-500 mu M) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K-d values for NAD(+) and NADH are similar to those previously reported with isolated dI, but the K-d values for NADP(+) and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidised. (c) 2005 Elsevier B.V. All rights reserved.
UR - http://www.scopus.com/inward/record.url?scp=21744448180&partnerID=8YFLogxK
U2 - 10.1016/j.bbabio.2005.04.004
DO - 10.1016/j.bbabio.2005.04.004
M3 - Article
C2 - 15935988
VL - 1708
SP - 404
EP - 410
JO - Biochimica et Biophysica Acta. Bioenergetics
JF - Biochimica et Biophysica Acta. Bioenergetics
IS - 3
ER -