Are there different water requirements in different steps of a catalytic cycle? Hydration effects at the E1 and E2 conformers of sarcoplasmic reticulum Ca(2+)-ATPase studied in organic solvents with low amounts of water.
The Ca(2+)-ATPase from sarcoplasmic reticulum was transferred in an active form to a low-water system composed of toluene, phospholipids, and Triton X-100 (TPT). The Ca(2+)-ATPase activity in the TPT system with 4.0% water (by vol. was about 50% of the activity observed in all-aqueous mixtures. Phosphate formation was linear with time up to 20% of ATP hydrolysis and, as expected from an enzyme-catalysed reaction, activity was linear with protein concentration. No ATPase activity was detected in the presence of 3 mM EGTA, indicating that the enzyme retained its Ca2+ dependence in the TPT system. A hyperbolic response to ATP concentration was observed with a Km of 0.15 mM. There was no detectable ATPase activity at water concentrations below 1.5% (by vol.). With 2.0% water, activity became detectable and increased as the water content was progressively raised to 7.0% (by vol.). Higher amounts of water produced unstable emulsions. Enzyme phosphorylation by ATP and dephosphorylation took place in the TPT system. The velocities of both enzyme phosphorylation and dephosphorylation increased with increments in the water content. The enzyme could also be phosphorylated in the TPT system by inorganic phosphate. However, in comparison to ATP, phosphorylation by phosphate took place with significantly lower amounts of water. It is suggested that at low amounts of water, the enzyme is in a relatively rigid conformation and, as the water content is increased, the ATPase acquires more flexibility and, hence, the capacity to carry out catalysis at higher rates. Nevertheless, the release of conformational constraints of the catalytic site of the E2 conformer takes place at water concentrations much lower than those needed for the expression of catalytic activity by the E1 conformer.
Barrabin
H
H
Departamento de Bioquímica, ICB, CCS, Universidade Federal, Rio de Janeiro, Brazil.
Scofano
H M
HM
de Gómez-Puyou
M T
MT
Gómez-Puyou
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D003902:Detergents; D007700:Kinetics; D017830:Octoxynol; D010710:Phosphates / Q000378:metabolism; D010743:Phospholipids; D011092:Polyethylene Glycols; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014050:Toluene
D003902:Detergents; D010710:Phosphates; D010743:Phospholipids; D014050:Toluene; D011092:Polyethylene Glycols; D000255:Adenosine Triphosphate; D017830:Octoxynol; D000252:Calcium-Transporting ATPases