The 3-D structure of yeast enolase (2-phospho-D-glycerate hydrolyase, E.C.4.2.1.11, m.w. of monomer 46,672) will be determined by X-ray crystallographic methods. The enzymatically active crystals scatter to 2.2 A resolution and very favorably the physiologically active dimer is demanded to lie on a crystallographic two fold axis of symmetry. Hence only 436 residues of known sequence have to be located. For the native and one heavy atom derivative (Sm3+) data up to 2.2A resolution have been measured using multiwire area X-ray diffractomer (at National Biotechnology Resource, Univ. of Virginia). 3.2A resolution data for a mercury derivative have measured using our conventional diffractometer. Each derivatives has one major heavy atom site, they are about 11A part. Enolase is the last major glycolytic enzyme whose structure is not known nor being actively investigated. Its folding will be compared with other glycoltic enzymes and the implications for the genesis of the glycolytic pathway and molecular evolution will be analyzed. The mechanism of enolytic catalysis will be investigated by crystal binding studies of substrate/product in the absence of the cofactor """"""""catalytic"""""""" Mg2+. Additional informtion will be obtained from binding transition state analogs with the cofactor present. Furthermore, the effects of various cations and anions activating and inhibiting enolase will be studied. Ca2+ binds but does not activate enolase so particular emphasis is put on Ca2+ enolase complex to study Ca/Mg specificity.
