STRUCTURAL AND THEORETICAL-STUDIES SUGGEST DOMAIN MOVEMENT PRODUCES AN ACTIVE CONFORMATION OF THYMIDINE PHOSPHORYLASE

Two new crystal forms of Escherichia coli thymidine phosphorylase (EC 2.4.2.4) have been found; a monoclinic form (space group P2(1)) and an orthorhombic form (space group I222). These structures have been solv ed and compared to the previously determined tetragonal. form (space g roup P4(3)2(1)2). This comparison provides evidence of domain movement of the alpha (residues 1 to 65, 163 to 193) and alpha/beta (residues 80 to 154, 197 to 440) domains, which is thought to be critical for en zymatic activity by closing the active site cleft. Three hinge regions apparently allow the alpha and alpha/beta-domains to move relative to each other. The monoclinic model is the most open of the three models while the tetragonal model is the most closed. Phosphate binding indu ces formation of a hydrogen bond between His119 and Gly208, which help s to order the 115 to 120 loop that is disordered prior to phosphate b inding. The formation of this hydrogen bond also appears to play a key role in the domain movement. The a-domain moves as a rigid body, whil e the alpha/beta-domain has some non-rigid body movement that is assoc iated with the formation of the His119-Gly208 hydrogen bond. The 8 Ang strom distance between the two substrates reported for the tetragonal form indicates that it is probably not in an active conformation. Howe ver, the structural data for these two new crystal forms suggest that closing the interdomain cleft around the substrates may generate a fun ctional active site. Molecular modeling and dynamics simulation techni ques have been used to generate a hypothetical closed conformation of the enzyme. Analysis of this model suggests several residues of possib le catalytic importance. The model explains observed kinetic results a nd satisfies requirements for efficient enzyme catalysis, most notably through the exclusion of water from the enzyme's active site. (C) 199 8 Academic Press.