Interactions of phosphate groups of ATP and aspartyl phosphate with the sarcoplasmic reticulum Ca²⁺-ATPase. An FTIR study

¹ Johann Wolfgang Goethe-Universität and Stockholm University
² Stockholm University

^* To whom correspondence should be addressed. E-mail: barth{at}dbb.su.se.

Submitted on February 23, 2005
Revised on April 13, 2005
Accepted on 23 August 2005

	Abstract

Phosphate binding to the sarcoplasmic reticulum Ca²⁺-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([-¹⁸O₂, -¹⁸O]ATP and [-¹⁸O₃]ATP). Isotopic substitution identified several bands which can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110 and 1085 cm^-1 are affected by labeling of the -phosphate, bands likely near 1154, and 1098-1089 cm^-1 by -phosphate labeling. The findings indicate that the strength of interactions of - and - phosphate with the protein are similar to those in aqueous solution. Two bands were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca₂E1P: at 1175 and 1113 cm^-1. They indicate terminal and bridging P-O bond strengths that are intermediate between those of E2P and the model compound acetyl phosphate in water. The bridging bond of Ca₂E1P is weaker than for acetyl phosphate which will facilitate phosphate transfer to ADP, but is stronger than for E2P which will make the Ca2E1P phosphate less susceptible to attack by water.

Key Words: ATP binding, SERCA1a, caged ATP, infrared spectroscopy, phosphoenzyme

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