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Membranes and Bioenergetics

Chemical Equivalence of Phosphoenzyme Reaction States in the Catalytic Mechanism of the Red Beet (Beta vulgaris L.) Plasma Membrane ATPase ¹

Department of Agronomy, University of Illinois, 1102 South Goodwin Avenue, Urbana, Illinois 61801

A comparison of two phosphoryl enzyme reaction states associated with the plasma membrane ATPase of red beet (Beta vulgaris L.) storage tissue was carried out to determine if their differences in reactivity toward ADP and K⁺ was related to a structural difference in the site of phosphorylation. Using a pulse labeling method it was possible to produce preparations where either the ADP-sensitive and -insensitive phosphoenzyme forms or the ADP-insensitive phosphoenzyme form alone were trapped as trichloroacetic acid denatured protein. Following complete digestion with Pronase, both preparations yielded radioactive tripeptides with similar properties with respect to pH stability of the covalent bond linking the phosphate to the peptide, isoelectric point, and migration on cellulose thin layer plates. Since the preparation containing both intermediate reaction states behaved in a uniform manner during analysis and displayed properties similar to the preparation containing only the ADP-insensitive phosphoenzyme form, it was proposed that both phosphoenzyme forms were chemically equivalent and derived from the same region of the catalytic active site. The observation that ethyleneimine treatment of both preparations followed by trypsin digestion resulted in the production of tripeptides similar to the Pronase fragments would support this proposal since it suggests that the tripeptides from both phosphoenyzme states contain a lysine residue on the C terminal end and are adjacent to a cysteine residue on the N-terminal end. The chemical equivalence of these two phosphoenzyme reaction states suggests that their differences in reactivity toward ligands may be related to conformational changes associated with the catalytic and transport mechanism of this enzyme.