The ATP synthase beta subunit hinge domain (betaPhe148 approximately betaGly186, P-loop/alpha-helixB/loop/beta-sheet4, Escherichia coli residue numbering) dramatically changes in conformation upon nucleotide binding. We previously reported that F(1) with the betaSer174 to Phe mutation in the domain lowered the gamma subunit rotation speed, and thus decreased the ATPase activity [M. Nakanishi-Matsui, S. Kashiwagi, T. Ubukata, A. Iwamoto-Kihara, Y. Wada, M. Futai, Rotational catalysis of Escherichia coli ATP synthase F(1) sector. Stochastic fluctuation and a key domain of the beta subunit, J. Biol. Chem. 282 (2007) 20698-20704.]. Homology modeling indicates that the amino acid replacement induces a hydrophobic network, in which the betaMet159, betaIle163, and betaAla167 residues of the beta subunit are involved together with the mutant betaPhe174. The network is expected to stabilize the conformation of beta(DP) (nucleotide-bound form of the beta subunit), resulting in increased activation energy for transition to beta(E) (empty beta subunit). The modeling further predicts that replacement of betaMet159 with Ala or Ile weakens the hydrophobic network. As expected, these two mutations experimentally suppressed the ATPase activities as well as subunit rotation of betaS174F. Furthermore, the rotation rate decreased with the increase of the strength in the hydrophobic network. These results indicate that the smooth conformational change of the beta subunit hinge domain is pertinent for the rotational catalysis.