A molecular coupling mechanism for the oxaloacetate decarboxylase Na+ pump as inferred from mutational analysis.
The oxaloacetate decarboxylase Na+ pump consists of subunits alpha, beta, and gamma, and contains biotin as the prosthetic group. Membrane-bound subunit beta catalyzes the decarboxylation of carboxybiotin coupled to Na+ translocation, and consumes a periplasmically derived proton. Site-directed mutagenesis of conserved amino acids of transmembrane helix VIII indicated that residues N373, G377, S382, and R389 are functionally important. The polar side groups of these amino acids may constitute together with D203 a network of ionizable groups which promotes the translocation of Na+ and the oppositely oriented H+ across the membrane. Evidence is presented that two Na+ ions are bound simultaneously to subunit beta during transport with D203 and S382 acting as binding sites. Sodium ion binding from the cytoplasm to both sites elicits decarboxylation of carboxybiotin, and a conformational switch exposes the bound Na+ ions toward the periplasm. After dissociation of Na+ and binding of H+, the cytoplasmically exposed conformation is regained.
Jockel
P
P
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland.
Schmid
M
M
Steuber
J
J
Dimroth
P
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001710:Biotin / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D002262:Carboxy-Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005822:Genetic Vectors / Q000138:chemical synthesis; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics*; D014357:Trypsin / Q000737:chemistry
D011994:Recombinant Proteins; C024582:N1'-carboxybiotin; D001710:Biotin; D012964:Sodium; D014357:Trypsin; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D000254:Sodium-Potassium-Exchanging ATPase