Solution structure and dynamics of an open β-sheet, glycolytic enzyme, monomeric 23.7 kDa phosphoglycerate mutase from Schizosaccharomyces pombe

Abstract

The structure and backbone dynamics of a double labelled (15N,13C) monomeric, 23.7 kD phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe have been investigated in solution using NMR spectroscopy. A set of 3125 NOE-derived distance restraints, 148 restraints representing inferred hydrogen bonds and 149 values of 3JHNHa were used in the structure calculation. The mean rmsd from the average structure for all backbone atoms from residues 6-205 in the best 21 calculated structures was 0.59 AÊ . The core of the enzyme includes an open, twisted, six-stranded b-sheet ¯anked by four a-helices and a short 310-helix. An additional smaller domain contains two short antiparallel b-strands and a further pair of a-helices. The Ca atoms of the S. pombe PGAM may be superimposed on their equivalents in one of the four identical subunits of Saccharomyces cerevisiae PGAM with an rmsd of 1.34 AÊ (0.92 AÊ if only the b-sheet is considered). Small differences between the two structures are attributable partly to the deletion in the S. pombe sequence of a 25 residue loop involved in stabilising the S. cerevisciae tetramer. Analysis of 15N relaxation parameters indicates that PGAM tumbles isotropically with a rotational correlation time of 8.7 ns and displays a range of dynamic features. Of 178 residues analysed, only 77 could be ®tted without invoking terms for fast internal motion or chemical exchange, and out of the remainder, 77 required a chemical exchange term. Signi®cantly, 46 of the slowly exchanging (milli- to microsecond) residues lie in helices, and these account for two-thirds of all analysed helix residues. On the contrary, only one b-sheet residue required an exchange term. In contrast to other analyses of backbone dynamics reported previously, residues in slow exchange appeared to correlate with architectural features of the enzyme rather than congregating close to ligand binding sites