Sequence, structure and activity of yeast 3-phosphoglycerate kinase
The four cyanogen bromide fragments of yeast 3-phosphoglycerate kinase (PGK) have been isolated and characterised. After digestion with proteolytic enzymes and specific cleavage reagents, the resuting peptides were purified by various methods and sequenced using the manual dansyl-Edman technique and the Beckman 890C liquid phase sequencer. The entire sequence of yeast PGK (415 residues) has been determined using a combination of amino acid sequence data and nucleotide sequence data. Nucleotide sequence data were supplied by Dr. A. Kingsman, University of Oxford. The yeast PGK sequence data have been fitted tothe 2.S electron density map and the nucleotide binding site has been fully characterised. The fitting of sequence data to the electron density map permitted identification of additional electron density which is probably attributable to the triose phosphate substrate. This binding site has also been characterised. The construction of the 1g:1cm model of yeast PGK permitted interpretation of chemical modification, NMR, hydrodynamic and kinetic data from a structural point of veiw, thereby allowing a catalytic mechanism to be proposed. This mechanism involves a major conformational change, triggered by the breaking of a salt-bridge between glutamate 190 and histidine 388 concommittant with the formation of the ternary enzyme-substrates complex. The conformational change brings the two substrates into close proximity, thereby permitting the in-line, direct, associative,phosphoryl transfer reaction to take place. The hydrodynamic properties of yeast PGK were examined in order to determine conditions under which PGK adopted its closed , catalytically active conformation. The solubility of yeast PGK in organic solvents commonly used as crystallising media was examined and experiments performed which were designed to crystallise a) the closed conformation of yeast PGK, and b) the substrate-free form of yeast PGK. No crystals have yet been observed in these experiments.