Thermodynamic analyses of nucleotide binding to an isolated monomeric ? subunit and the ?3?3? subcomplex of F1-ATPase.

ABSTRACT: Rotation of the ? subunit of the F1-ATPase plays an essential role in energy transduction by F1-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F1-ATPase. In this study, we introduced a ?Y341W mutation as an optical probe for nucleotide binding to catalytic sites, and a ?E190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric ?Y341W subunit and a mutant ?3?3? subcomplex containing the ?Y341W mutation with or without an additional ?E190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/Kd) of the nucleotides for the isolated ? subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van't Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated ? subunit, NDPs and NTPs with the same base moiety exhibited similar ?H(0) and ?G(0) values at 25°C. The binding of nucleotides with different bases to the isolated ? subunit resulted in different entropy changes. Interestingly, NDP binding to the ?3?(Y341W)3? subcomplex had similar Kd and ?G(0) values as binding to the isolated ?(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.

SUBMITTER: Kikuchi Y 

PROVIDER: S-EPMC3853085 | biostudies-other | 2013 Dec

REPOSITORIES: biostudies-other