Time-resolved infrared spectroscopy of the Ca2+-ATPase. The enzyme at work.
Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca2+-ATPase in the course of its catalytic cycle were followed in real time using rapid scan Fourier transform infrared spectroscopy. In the presence of Ca2+, the cycle was induced by the photochemical release of ATP from a biologically inactive precursor (caged ATP, P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate). Absorbance changes arising from ATP binding to the ATPase were observed within the first 65 ms after initiation of ATP release. After ATP binding, up to two subsequent partial reactions of the ATPase reaction cycle were observed depending on the buffer composition (10 mM CaCl2 + 330 mM KCl or 1 mM CaCl2 + 20% Me2SO): (i) formation of the ADP-sensitive phosphoenzyme (kapp = 0.79 s-1 +/- 15% at 1 degrees C, pH 7.0, 10 mM CaCl2, 330 mM KCl) and (ii) phosphoenzyme conversion to the ADP-insensitive phosphoenzyme concomitant with Ca2+ release (kapp = 0.092 s-1 +/- 7% at 1 degrees C, pH 7.0, 1 mM CaCl2, 20% Me2SO). Each reaction step could well be described by a single time constant for all associated changes in the vibrational spectrum, and no intermediates other than those mentioned above were found. In particular, there is no evidence for a delay between the transition from ADP-sensitive to ADP-insensitive phosphoenzyme and Ca2+ release. In 2H2O a kinetic isotope effect was observed: both the phosphorylation reaction and phosphoenzyme conversion were slowed down by factors of 1.5 and 3.0, respectively. The small amplitudes of the observed changes in the infrared spectrum indicate that the net change of secondary structure is very small and of the same order of magnitude for ATP binding, phosphorylation, and phosphoenzyme conversion. Therefore, our results do not support a distinction between minor and major secondary structure changes in the catalytic cycle of the ATPase, which might be expected according to the classical E1-E2 model.
Barth
A
A
Institut für Biophysik und Strahlenbiologie der Universität Freiburg, Albertstrasse 23, D-79104 Freiburg, Germany.
von Germar
F
F
Kreutz
W
W
Mäntele
W
W
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D002474:Cell-Free System; D009038:Motion; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared
D010750:Phosphoproteins; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases