Time-resolved Infrared Spectroscopy of the Ca2+-ATPase

Abstract

Changes in the vibrational spectrum of the sarcoplasmic reticulum Ca 2؉ -ATPase in the course of its catalytic cycle were followed in real time using rapid scan Fourier transform infrared spectroscopy. In the presence of Ca 2؉ , the cycle was induced by the photochemical release of ATP from a biologically inactive precursor (caged ATP, P 3 -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 CaCl 2 ؉ 330 mM KCl or 1 mM CaCl 2 ؉ 20% Me 2 SO): (i) formation of the ADP-sensitive phosphoenzyme (k app ‫؍‬ 0.79 s ؊1 ؎ 15% at 1°C, pH 7.0, 10 mM CaCl 2 , 330 mM KCl) and (ii) phosphoenzyme conversion to the ADP-insensitive phosphoenzyme concomitant with Ca 2؉ release (k app ‫؍‬ 0.092 s ؊1 ؎ 7% at 1°C, pH 7.0, 1 mM CaCl 2 , 20% Me 2 SO). 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 Ca 2؉ release. In 2 H 2 O 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 E 1 -E 2 model. -transport across the SR membrane to the hydrolysis of ATP. Its reaction cycle is shown in a simplified form in Fig. 1. Ca 2ϩ is bound from the cytoplasmic side of the membrane to high affinity binding sites of the ATPase (step on the left of Fig. 1), which enables the ATPase to use ATP as a substrate (1). Phosphorylation by ATP (upper step in Fig. 1) results in the occlusion of the bound Ca 2ϩ in the protein. The subsequent conversion of the phosphoenzyme from the ADP-sensitive to the ADP-insensitive form (step on the right of Fig. 1) leads to Ca 2ϩ release into the SR lumen. Hydrolytic cleavage of the phosphoenzyme completes the reaction cycle (bottom step in Fig. 1). An ATP molecule is shown bound to the ATPase throughout the cycle, which is the case at millimolar ATP concentrations (reviewed in Ref.2).The original model of the reaction cycle from deMeis and Vianna (3) was based on the assumption of two main functional states, E 1 and E 2 , of the protein. The interconversion between E 1 and E 2 is thought to be associated with a reorientation of the Ca 2ϩ -binding sites from the cytoplasmic s...

Mapping Interactions between the Ca2+-ATPase and Its Substrate ATP with Infrared Spectroscopy

“…Replacement of Mg 2ϩ at the catalytic site by Ca 2ϩ decreases the rate of phosphorylation by 1 order of magnitude (30 -32), enabling a longer observation time for E1ATPCa 2 and therefore a better signal to noise ratio of the ATP binding spectrum. Ca 2ϩ instead of Mg 2ϩ has been used by us before (29,33,34) and gave very similar spectra for the E1Ca 2 3 E1PCa 2 (34) …”

“…These conditions were chosen to block the E1PCa 2 3 E2P transition to achieve a maximum level of E1PCa 2 in the steady state after nucleotide release while slowing down the phosphorylation reaction (29). Replacement of Mg 2ϩ at the catalytic site by Ca 2ϩ decreases the rate of phosphorylation by 1 order of magnitude (30 -32), enabling a longer observation time for E1ATPCa 2 and therefore a better signal to noise ratio of the ATP binding spectrum.…”

“…FTIR Measurements-Time-resolved Fourier transfer IR measurements of the Ca 2ϩ -ATPase reaction were performed at 1°C as described previously (28,29). Photolytic release of nucleotides from their respective caged derivatives was triggered by a xenon flash tube or a XeCl excimer laser.…”

“…ATPase phosphorylation were obtained by fitting the integrated band intensities of the marker band at 1628 cm Ϫ1 for nucleotide binding and two marker bands at 1721 and 1549 cm Ϫ1 for phosphorylation (29,35) with the second (the third for ITP results) and first order exponential decay equations, respectively (Origin 5.0), and averaging the resulting time constants. 23 experiments were averaged for ATP, 8 for 2Ј-deoxy-ATP and 11 for ITP.…”

Topology of sarcoplasmic reticulum Ca2+‐ATPase: An infrared study of thermal denaturation and limited proteolysis

“…IR has been used with Ca'+-ATPase to characterize the E , + E2 conformational transition either by studying the amide I band (Arrondo et al, 1987;Villalain et al, 1989) or by using caged compounds (Buchet et al, 1991;Barth et al, 1994). Recently, the kinetic mechanism of the enzyme has been described by time-resolved difference spectroscopy using ATP (Barth et al, 1996). Also, IR has been used to characterize the proteolytic digestion of the nicotinic acetylcholine receptor (Come-Tschelnokow et al, 1994) and P-type ATPases such as the Neurospora Hf-ATPase (Vigneron et al, 1995) or the Ca'+-ATPase from sarcoplasmic reticulum (Corbalan-Garcia et al, 1994).…”