TY - JOUR
T1 - Mg2+ coordination in catalytic sites of F1-ATPase
AU - Weber, Joachim
AU - Hammond, Sean T.
AU - Wilke-Mounts, Susan
AU - Senior, Alan E.
N1 - Copyright:
Copyright 2004 Elsevier Science B.V., Amsterdam. All rights reserved.
PY - 1998/1/13
Y1 - 1998/1/13
N2 - Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli F1-ATPase was investigated. From the X-ray structure of the mitochondrial enzyme [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], it may be inferred that the hydroxyl of βThr-156 is a direct ligand of Mg2+, whereas the carboxyls of βGlu-181, βGlu-185, and βAsp- 242 might contribute via intervening water molecules. Elimination of each respective functional group by site-directed mutagenesis, followed by determination of Mg-nucleotide and uncomplexed nucleotide binding affinities using a tryptophan probe, showed that βThr-156, βGlu-185, and βAsp-242 are all involved in Mg2+ coordination, whereas βGlu-181 is not. A derived structural model for the octahedral coordination around the Mg2+ ion is presented. The results indicate that the ADP-containing site in the X-ray structure is the catalytic site of highest affinity. Correct Mg2+ coordination is required for catalytic activity at physiological rates. Elimination of any one of the Mg2+-coordinating residues led to complete loss of Mg2+-dependent nucleotide binding cooperativity of the catalytic sites.
AB - Coordination of the Mg2+ ion in Mg-nucleotide substrates by amino acid residue side chains in the catalytic site of Escherichia coli F1-ATPase was investigated. From the X-ray structure of the mitochondrial enzyme [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628], it may be inferred that the hydroxyl of βThr-156 is a direct ligand of Mg2+, whereas the carboxyls of βGlu-181, βGlu-185, and βAsp- 242 might contribute via intervening water molecules. Elimination of each respective functional group by site-directed mutagenesis, followed by determination of Mg-nucleotide and uncomplexed nucleotide binding affinities using a tryptophan probe, showed that βThr-156, βGlu-185, and βAsp-242 are all involved in Mg2+ coordination, whereas βGlu-181 is not. A derived structural model for the octahedral coordination around the Mg2+ ion is presented. The results indicate that the ADP-containing site in the X-ray structure is the catalytic site of highest affinity. Correct Mg2+ coordination is required for catalytic activity at physiological rates. Elimination of any one of the Mg2+-coordinating residues led to complete loss of Mg2+-dependent nucleotide binding cooperativity of the catalytic sites.
UR - http://www.scopus.com/inward/record.url?scp=0032512370&partnerID=8YFLogxK
U2 - 10.1021/bi972370e
DO - 10.1021/bi972370e
M3 - Article
C2 - 9425083
AN - SCOPUS:0032512370
VL - 37
SP - 608
EP - 614
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 2
ER -