Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study.

Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: functional and structural considerations.

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca(2+) ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca2+ binding site are thereby changed, permitting vectorial dissociation of bound Ca2+ against a concentration gradient. A long range linkage between phosphorylation and Ca2+ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.

Intracellular signaling through long-range linked functions in the Ca2+ transport ATPase.

The Ca2+ transport ATPases of intracellular membranes exhibit an intracellular long-range functional linkage which is the basic mechanistic device for Ca2+ transport through ATP utilization. The functional linkage operates between a phosphorylation (catalytic) domain located in the extramembranous region, and a Ca2+ binding domain located in the membrane bound region of the enzyme. The two domains are separated by a distance of approximately 50 A, and are both affected by binding of a single molecule of the highly specific inhibitor, thapsigargin, to the enzyme. Functional and structural features are here described to explain the long-range linkage through the protein structure.

Role of lysine residues in the binding of glyceraldehyde-3-phosphate dehydrogenase to human erythrocyte membranes.

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) binds reversibly to human erythrocyte membranes. Several specific amino acid residues involved in the enzyme-membrane contact region have already been identified. These include tyrosine 46 and threonine 150. Covalent modification of lysines 212 and 191 with pyridoxal phosphate results in a decreased affinity of the enzyme for erythrocyte membranes if the enzyme-linked pyridoxal phosphate is not reduced prior to binding. Reduction of the pyridoxal phosphate-lysine complex completely inhibits the binding of the enzyme to erythrocyte membranes. These results suggest a role for lysines 212 and 191 in the interaction of glyceraldehyde-3-phosphate with human erythrocyte membranes.

Fructose-1,6-bisphosphate, a regulator of metabolism.

Fructose-1,6-bisphosphate affects the rate of a large variety of enzyme reactions. In some instances its role as a physiologic effector is well documented. In many cases the effects of fructose bishosphate on particular enzymes have been demonstrated in vitro but the link to physiologic conditions has not yet been established. It is the purpose of this paper to summarize the scattered findings in fructose bisphosphate as an effector of enzyme reactions and to draw some conclusions about the role of the compound in metabolic regulation.

Interaction of phosphoglycerate kinase with human erythrocyte membranes.

The purpose of this study was to investigate the interaction of phosphoglycerate kinase with the human erythrocyte ghost membrane. Ghosts prepared in 0.1 mM EDTA and 17 mM Tris buffer (pH 7.5) have about 230 molecules of phosphoglycerate kinase/ghost. No additional binding is observed after incubating soluble enzyme with these leaky ghosts. This binding is tight but reversible with Kd = 7.1 X 10(-10) M. The enzyme can be eluted significantly from the membrane by incubation with 0.15 M NaCl and it rebinds to the membrane when the depleted ghosts are incubated with rabbit muscle phosphoglycerate kinase. Ligand binding studies show that NADH and NAD have opposite effects on the binding of the enzyme to the membrane; NAD (1.0 MM) favors binding while NADH (0.25 MM) does not. Similarly, ADP (0.2 mM) favors binding while ATP does not. ATP elutes the membrane-bound enzyme with Kd = 0.058 mM. MgSO4 also stimulates dissociation of the membrane-bound phosphoglycerate kinase (Kd = 0.36 mM), an effect which appears to be due to the magnesium ion. ADP (0.2 mM) can counteract the negative effect of MgSO4 (1.0 mM) on binding of phosphoglycerate kinase to the membrane. We have been unable so far to find tight coupling of the (Na+-K+)-ATPase with the membrane-bound phosphoglycerate kinase.