Sensitivity of spin-labeled sarcoplasmic reticulum to the phosphorylation state of the catalytic site in aqueous media and in dimethyl sulfoxide.

Under controlled conditions, the iodoacetamide spin-label is highly selective for the sarcoplasmic reticulum adenosinetriphosphatase (SR ATPase), labeling 7-8 nmol of reactive residues per mg of SR protein, or approximately two residues per active enzyme unit. The electron paramagnetic resonance (EPR) spectrum of the labeled enzyme exhibits a small but specific broadening on the binding of substrates or inorganic phosphate. Addition of Ca2+ greatly increases the substrate broadening but reverses the effect of Pi. Here we demonstrate that the Ca2+ effect on the enzyme-substrate spectrum is due to the division of the major spectral component into two components, each representing two distinct conformational environments which impart slightly different degrees of mobility to the spin-label. These components are not readily separated in the spectrum of the fully labeled enzyme, and stoichiometric labeling techniques are used to resolve the components and obtain splitting parameters. The two spectral components exhibit random redistribution on removal and reintroduction of substrate, indicating that they may represent two forms of a given site. E-P formation by Pi in the absence of Ca2+ does not produce two discernible components in the EPR spectrum, although a small broadening effect is apparent, and two components can be resolved in the reaction kinetics of N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide with SR. However, addition of dimethyl sulfoxide, which greatly facilitates phosphorylation of the enzyme, induces a two-component EPR spectrum. We conclude that a conformational change occurs in the enzyme specifically under conditions of high phosphate affinity which affects the motional parameters of 3-4 nmol of labeled residues per mg of SR, a number equal to the phosphorylation sites in our preparations. This affinity is induced by substrate and Ca2+ binding at activating sites in the normal mode of the enzymatic cycle, or by dimethyl sulfoxide in a general manner. In addition, we observe the conformational change with non-adenosine substrates in varying degrees, while 5'-adenylyl imidodiphosphate and adenosine 5'-(beta, gamma-methylenetriphosphate) are both fully effective. Since the latter analogues do not actively phosphorylate the enzyme, it is concluded that the conformational change is related to formation of a transition complex which is highly permissive of phosphoryl transfer. This complex is maximized for an adenosine moiety, while Ca2+ remains a stringent requirement.