STRUCTURE OF RABBIT MUSCLE PYRUVATE-KINASE COMPLEXED WITH MN2+, K+, AND PYRUVATE

The molecular structure of rabbit muscle pyruvate kinase, crystallized as a complex with Mn2+, K+, and pyruvate, has been solved to 2.9-Angs trom resolution. Crystals employed in the investigation belonged to th e space group P1 and had unit cell dimensions a = 83.6 Angstrom, b = 1 09.9 Angstrom, c 146.8 Angstrom, alpha = 94.9 degrees, beta = 93.6 deg rees, and gamma = 112.3 degrees. There were two tetramers in the asymm etric unit. The structure was solved by molecular replacement, using a s 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., and Schmitt, W. (1986) E MBOJ. 5, 475-481]. The amino acid sequence derived from the cDNA codin g for the enzyme from rabbit muscle was fit to the electron density. T he rabbit and cat muscle enzymes have similar to 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 an d rabbit pyruvate kinases differ. Mn2+ coordinates to the protein thro ugh the carboxylate side chains of Glu 271 and Asp 295. These two resi dues are strictly conserved in all known pyruvate kinases. In addition , the density for Mn2+ is connected to that of pyruvate, consistent wi th chelation through a carboxylate oxygen and the carbonyl oxygen of t he 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 fo ur oxygen ligands contributed by the carbonyl oxygen of Thr 113, O-gam ma of Ser 76, O-delta 1 of Asn 74, and O-delta 2 of Asp 112. The inter action of Asp 112 with the side chains of Lys 269 and Arg 72 may media te, indirectly, monovalent cation effects on activity.