STRUCTURE OF THE BIS(MG2-ATP-OXALATE COMPLEX OF THE RABBIT MUSCLE PYRUVATE-KINASE AT 2.1 ANGSTROM RESOLUTION - ATP BINDING OVER A BARREL())

Pyruvate kinase from rabbit muscle has been cocrystallized as a comple x with Mg(II)ATP, oxalate, Mg2+, and either K+ or Na+, Crystals with e ither Na+ or K+ belong to the space group P2(1)2(1)2(1), and the asymm etric units contain two tetramers. The structures were solved by molec ular replacement and refined to 2.1 (K+) and 2.35 Angstrom (Na+) resol ution. The structures of the Na+ and K+ complexes are virtually isomor phous. Each of the eight subunits within the asymmetric unit contains Mg(II)oxalate as a bidentate complex linked to the protein through coo rdination of Mg2+ to the carboxylates of Glu 271 and Asp 295. Six of t he subunits also contain an alpha,beta,gamma-tridentate complex of Mg( II)ATP, and the active-site cleft, located between domains A and B, is closed in these subunits. In the remaining two subunits Mg(II)ATP 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 degree s of the B domain relative to the A domain. alpha-Carbons of residues in the B domain undergo movements of up to 17.8 Angstrom (Lys 124) in the cleft closure, Lys 206, Arg 119, and Asp 177 from the B domain mov e several angstroms from their positions in the open conformation to c ontact the Mg(II)ATP complex in the active site. The gamma-phosphate o f ATP coordinates to both magnesium ions and to the monovalent cation, K+ or Na+. A Mg2+-coordinated oxygen from the Mg(II)oxalate complex l ies 3.0 Angstrom from Py of ATP, and this oxygen is positioned for an in-line attack on the phosphorus. The side chains of Lys 269 and Arg 1 19 are positioned to provide leaving-group activation in the forward a nd 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 hy droxyls may function in a proton relay.