CRYO-CRYSTALLOGRAPHY OF A TRUE SUBSTRATE, INDOLE-3-GLYCEROL PHOSPHATE, BOUND TO A MUTANT (ALPHA-D60N) TRYPTOPHAN SYNTHASE ALPHA(2)BETA(2) COMPLEX REVEALS THE CORRECT ORIENTATION OF ACTIVE-SITE ALPHA-GLU49

The reversible cleavage of indole-3-glycerol by the cy-subunit of tryp tophan synthase has been proposed to be catalyzed by alpha Glu49 and a lpha Asp60. Although previous x-ray crystallographic structures of the tryptophan synthase alpha(2) beta(2) complex showed an interaction be tween the carboxylate of alpha Asp60 and the bound inhibitor indole-3- propanol phosphate, the carboxylate of alpha Glu49 was too distant to play its proposed role, To clarify the structural and functional roles of alpha Glu49, we have determined crystal structures of a mutant (al pha D60N) alpha(2) beta(2) complex in the presence and absence of the true substrate, indole-3-glycerol phosphate. The enzyme in the crystal cleaves indole-3-glycerol phosphate very slowly at room temperature b ut not under cryo-conditions of 95 K. The structure of the complex wit h the true substrate obtained by cryo-crystallography reveals that ind ole-3-glycerol phosphate and indole-3-propanol phosphate have similar binding modes but different torsion angles. Most importantly, the side chain of alpha Glu49 interacts with S-hydroxyl group of indole-3-glyc erol phosphate as proposed, The movement of the side chain of alpha Gl u49 into an extended conformation upon binding the true substrate prov ides evidence for an induced fit mechanism. Our results demonstrate ho w cryo-crystallography and mutagenesis can provide insight into enzyme mechanism.