Structure determination and refinment of <i>Bacillus stearothermophilus</i> lactate dehydrogenase

<jats:title>Abstract</jats:title><jats:p>Structures have been determined of <jats:italic>Bacillus stearothermophilus</jats:italic> “apo” and holo lactate dehydrogenase. The holo‐enzyme had been co‐crystallized with the activator fructose 1,6‐biosphosphate. The “apo” lactate dehydrogenase structure was solved by use of the known apo‐M<jats:sub>4</jats:sub> dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 Å resolution by means of the noncrystallographic molecular 222 symmetry. The <jats:italic>R</jats:italic>‐factor was reduced to 28.7%, using 2.8 Å resolution data, in a restrained least‐squares refnement in which the molecula rsymmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the “apo” enzyme.</jats:p><jats:p>Further refinement proceeded with the isomorphous holo‐enzyume from <jats:italic>Bacillus Stearothermophilus</jats:italic>. After removing the noncrystallographic constraints, the <jats:italic>R</jats:italic>‐factor dropped from 30.3% to a final value of 26.0% with a 0.019 Å and 1.7° r.m.s. deviation from idealized bond length and angles, respectively.</jats:p><jats:p>Two sulfate ions per subunit were included in the final model of the “apo” ‐from‐one at the substrate binding site and one close to the molecular <jats:italic>P</jats:italic> ‐axis near the location of the fructose 1,6‐bisphosphate activator. The final model of the holo‐enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the <jats:italic>R</jats:italic>‐axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6‐bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6‐bisphosphate are close to the sulfate ion near the <jats:italic>P</jats:italic>‐axis in the “apo” model.</jats:p><jats:p>This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo‐enzyme showed far greater similarity to the ternary complex of dogfish M<jats:sub>4</jats:sub> lactate dehydrogenase with incotinamide adenine dinucleotide and oxamate than to apo‐M<jats:sub>4</jats:sub> dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose, 1,6‐bisphosphate were also analyzed.</jats:p>