P-type ATPases form a
large protein family with members in practically all cell types from
the archaea to humans. They generate essential ion gradients which
are the basis for such diverse functions like signalling, energy
storage and secondary transport processes. The name P-type for this
class of ATPases stems from the fact that they form a phosphorylated
intermediate during transport.
More than one third of the ATP
consumed by a resting animal is used by a single ion pump, the
Na+,K+-ATPase
which shows the significance of the transport processes mediated by
P-type ATPases. For the discovery of this ion pump and for his
further contributions the nobel prize 1997 was awarded to Jens C.
Skou.
In the following, a prominent member of the P-type
ATPase family is discussed in more detail.
The reason why you are
sitting relaxed in front of your computer screen is a molecular ion
pump in the sarcoplasmic reticulum (SR) membrane - the calcium
ATPase. This enzyme pumps calcium from the muscle cell into an
internal calcium store (SR) and consumes the fuel ATP. In consequence
the muscle relaxes.
The pump process proceeds in a reaction
cycle. Here the ordered sequence of the partial reactions ensures the
high efficiency of the pump, which means that there is no calcium
leak and no ATP hydrolysis without pumping. The scheme below shows a
simplified version of the reaction cycle.
Let´s start the
description of the cycle with the calcium free ATPase E (left hand
side, bottom). Two calcium ions bind from the cytoplasm of muscle
cells to the high affinity binding sites of the ATPase (left hand
side, top, only one calcium is shown in the scheme above). This
activates the ATPase to cleave the fuel molecule ATP. Thereby a
phosphate group is transfered to the ATPase and the two calcium ions
are occluded in the protein (right hand side, top). The following
step releases calcium to the lumen from low affinity binding sites
(right hand side, bottom). Later, the ATPase-phospate bond is
hydrolysed.
The controlled procedure of the reaction cycle is
ensured by a coupling between the calcium binding sites and the
phosphorylation site. Interestingly these 2 sites are far apart (see
structure).
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To illucidate the
molecular transport mechanism we use a special technique of infrared
difference spectroscopy which is based on the photolytic release of
molecules from inactive precursors, i.e. caged compounds (see
"Infrared Spectroscopy with Caged
Compounds"). This renders possible the detection of very
small absorbance changes against a high background absorbance with a
sensitivity high enough to detect the absorbance of single amino acid
residues in this protein of 1000 residues.
RESULTS
-
The changes of net secondary structure are surprisingly small
- A
distinction between major and minor conformational changes does not
seem to be justified.
- Structural changes induced by calcium
binding are reversed when calcium is released from the
phosphoenzyme
- The calcium ions are not transported when ATP
phosphorylates the enzyme
- Calcium binding site is accessible to
protons from both sides of the membrane in successive enzyme states
-
The environment of single functional groups has been
characterised
For more information on our work see "Gallery
of ATPase Spectra" or a recent review in FEBS Lett. 477
(2000) 151-156 Full
html version / pdf
version via FEBS Lett. home page
METHODS
-Time-resolved Fourier Transform Infrared Spectroscopy
-
Fluorescence
- Spectroscopy in the visible spectral range
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