The role of specific beta-gamma subunit interactions in oxyanion stimulation of the MgATP hydrolysis of a hybrid photosynthetic F1-ATPase.
Pairs of cysteine residues were introduced into the twisted N- and C-terminal helices of the gamma subunit of the chloroplast F1-ATPase to test, via disulfide cross-linking, potential inter-helical movements involved in catalysis of ATP hydrolysis. The extent of disulfide cross-linking was determined by estimating the amount of free sulfhydryl available for labeling with fluoresceinyl maleimide before and after cross-linking. Significant disulfide formation (50-75%) was observed between cysteines introduced at positions 30 and 31 in the N-terminal helix and 276 and 278 in the C-terminal helix. Cross-linking had no apparent effect on catalysis, therefore eliminating the involvement of large-scale inter-helical movements within this region of the gamma subunit in cooperative ATP hydrolysis. However, the presence of the two cysteines together in the gammaV31C/A276C double mutant, irrespective of whether or not they were cross-linked together, lowered the MgATPase activity by more than 70% and completely eliminated the well-known activating effect of the oxyanion sulfite. The CaATPase activity was unaffected. Similar but less pronounced effects were seen with the gammaK30C/A276C double mutant. The results indicate that residues at or near positions 31 and 276 within the twisted helical pair of the gamma subunit are required to overcome Mg2+ inhibition of ATP hydrolysis. These residues are likely to be involved in forming a point of contact between the gamma and beta subunits that is responsible for this effect.
Samra
Hardeep S
HS
Department of Molecular Biosciences, The University of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045, USA.
He
Feng
F
Degner
Nicholas R
NR
Richter
Mark L
ML
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D000838:Anions; D002736:Chloroplasts / Q000737:chemistry*; D004789:Enzyme Activation; D006868:Hydrolysis; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000648:ultrastructure; D010100:Oxygen / Q000737:chemistry*; D010788:Photosynthesis / Q000502:physiology; D011485:Protein Binding; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000648:ultrastructure
D000838:Anions; D020409:Molecular Motor Proteins; D021122:Protein Subunits; C005147:5'-adenylyl (beta,gamma-methylene)diphosphonate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D010100:Oxygen