Structure of Rabbit Muscle Pyruvate Kinase Complexed with Mn2+, K+, and Pyruvate

Abstract

The molecular structure of rabbit muscle pyruvate kinase, crystallized as a complex with Mn2+, K+, and pyruvate, has been solved to 2.9-A resolution. Crystals employed in the investigation belonged to the space group P1 and had unit cell dimensions a = 83.6 A, b = 109.9 A, c = 146.8 A, alpha = 94.9 degrees, beta = 93.6 degrees, and gamma = 112.3 degrees. There were two tetramers in the asymmetric unit. The structure was solved by molecular replacement, using as the search model the coordinates of the tetramer of pyruvate kinase from cat muscle [Muirhead, H., Claydon, D. A., Barford, D., Lorimer, C. G., Fothergill-Gilmore, L. A., Schiltz, E., & Schmitt, W. (1986) EMBO J.5, 475-481]. The amino acid sequence derived from the cDNA coding for the enzyme from rabbit muscle was fit to the electron density. The rabbit and cat muscle enzymes have approximately 94% sequence identity, and the folding patterns are expected to be nearly identical. There are, however, three regions where the topological models of the cat and rabbit pyruvate kinases differ. Mn2+ coordinates to the protein through the carboxylate side chains of Glu 271 and Asp 295. These two residues are strictly conserved in all known pyruvate kinases. In addition, the density for Mn2+ is connected to that of pyruvate, consistent with chelation through a carboxylate oxygen and the carbonyl oxygen of the substrate. The epsilon-NH2 of Lys 269 and the OH of Thr 327 lie on either side of the methyl group of bound pyruvate. Spherical electron density, assigned to K+, is located within a well-defined pocket of four oxygen ligands contributed by the carbonyl oxygen of Thr 113, O gamma of Ser 76, O delta 1 of Asn 74, and O delta 2 of Asp 112. The interaction of Asp 112 with the side chains of Lys 269 and Arg 72 may mediate, indirectly, monovalent cation effects on activity.

The allosteric regulation of pyruvate kinase

“…Analysis of the numerous X-ray diffraction data sets collected during structure determination has revealed that the B-domain varies its orientation from crystal to crystal, thereby causing non-isomorphism. In this respect, variability of the B domain orientation has been observed also in the cat [6] and rabbit [7] M1 PK structures, further emphasising the enzyme flexibility and modularity, two key properties which are at the heart of the regulatory mechanism. Moreover, these independent observations suggest that the T-and R-states probably represent, in PK, an ensemble of conformations rather than a single well defined structure.…”

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

“…Interactions such as this, between the monovalent ion and a hydroxyl, seem to be unique to b-galactosidases. The functions of monovalent cations at the active sites of all other enzymes that are known to have monovalent metal requirements [44][45][46][47][48][49] either help neutralize the negative charge of substrate phosphate groups, often in conjunction with a divalent cation, or they have structural importance, but in this case, Na þ interacts with a hydroxyl group.…”

Fifteen novel mutations inPKLRassociated with pyruvate kinase (PK) deficiency: Structural implications of amino acid substitutions in PK

“…The highly conserved Ala154 is located close to active site residues Arg116 (involved in substrate binding [Valentini et al, 2002] and ATP/ADP binding [Larsen et al, 1994;Muirhead et al, 1986;Rigden et al, 1999]) and Asp156 (involved in K 1 binding [Jurica et al, 1998;Larsen et al, 1994;Rigden et al, 1999;Wooll et al, 2001]). The approximate 50% decrease in PK enzymatic activity as measured in the heterozygous Patient 24 indicates that substitution with threonine may perturb this interaction, thereby hampering catalysis.…”