Structure of the bis(Mg2+)-ATP-oxalate complex of the rabbit muscle pyruvate kinase at 2.1 A resolution: ATP binding over a barrel.
Pyruvate kinase from rabbit muscle has been cocrystallized as a complex with MgIIATP, oxalate, Mg2+, and either K+ or Na+. Crystals with either Na+ or K+ belong to the space group P2(1)2(1)2(1), and the asymmetric units contain two tetramers. The structures were solved by molecular replacement and refined to 2.1 (K+) and 2.35 A (Na+) resolution. The structures of the Na+ and K+ complexes are virtually isomorphous. Each of the eight subunits within the asymmetric unit contains MgIIoxalate as a bidentate complex linked to the protein through coordination of Mg2+ to the carboxylates of Glu 271 and Asp 295. Six of the subunits also contain an alpha,beta,gamma-tridentate complex of MgIIATP, and the active-site cleft, located between domains A and B, is closed in these subunits. In the remaining two subunits MgIIATP is missing, and the active-site cleft is open. Closure of the active-site cleft in the fully liganded subunits includes a rotation of 41 degrees of the B domain relative to the A domain. alpha-Carbons of residues in the B domain undergo movements of up to 17.8 A (Lys 124) in the cleft closure. Lys 206, Arg 119, and Asp 177 from the B domain move several angstroms from their positions in the open conformation to contact the MgIIATP complex in the active site. The gamma-phosphate of ATP coordinates to both magnesium ions and to the monovalent cation, K+ or Na+. A Mg2+-coordinated oxygen from the MgIIoxalate complex lies 3.0 A from Pgamma of ATP, and this oxygen is positioned for an in-line attack on the phosphorus. The side chains of Lys 269 and Arg 119 are positioned to provide leaving-group activation in the forward and reverse directions. There is no obvious candidate for the acid/base catalyst near the 2-si face of the prospective enolate of the normal substrate. A functional group linked through solvent and side-chain hydroxyls may function in a proton relay.
Larsen
T M
TM
Institute for Enzyme Research, Graduate School, and Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA.
Benning
M M
MM
Rayment
I
I
Reed
G H
GH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010070:Oxalates / Q000378:metabolism*; D019815:Oxalic Acid; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits; D012964:Sodium / Q000378:metabolism
D010070:Oxalates; D008345:Manganese; D000255:Adenosine Triphosphate; D019815:Oxalic Acid; D012964:Sodium; D011770:Pyruvate Kinase; D008274:Magnesium; D011188:Potassium