A BISUBSTRATE ANALOG INDUCES UNEXPECTED CONFORMATIONAL-CHANGES IN PHOSPHOGLYCERATE KINASE FROM TRYPANOSOMA-BRUCEI

The glycolytic enzyme phosphoglycerate kinase (PGK) catalyzes phosphor yl transfer between 1,3-bis-phosphoglycerate and ADP to form 3-phospho glycerate and ATP. During catalysis, a major hinge bending motion occu rs which brings the N and C-terminal enzyme domains and their bound su bstrates together and in-line for phosphoryl transfer. We have crystal lized Trypanosoma brucei PGK in the presence of the bisubstrate analog , adenylyl 1,1,5,5-tetrafluoropentane-1,5-bisphosphonate, and solved t he structure of this complex in two different crystal forms at 1.6 and 2.0 Angstrom resolution, obtained from PEG 8000 and ammonium phosphat e solutions, respectively. These high resolution structures of PGK:inh ibitor complexes are of particular interest for drug design since Tryp anosoma brucei, the causative agent of African sleeping sickness, reli es on glycolysis as its sole energy source. In both structures, the in hibitor is bound in a fully extended conformation with its adenosine m oiety assuming exactly the same position as in ADP:PGK complexes and w ith its 5' phosphonate group occupying part of the 1,3-bis-phosphoglyc erate binding site. The bisubstrate analog forces PGK to assume a nove l, ''inhibited'' conformation, intermediate in hinge angle between the native structures of open and closed form PGK. These structures of en zyme-inhibitor complexes demonstrate that PGK has two distinct hinge p oints that can each be independently activated. In the ''PEG'' structu re, the C-terminal hinge is partially activated while the N-terminal h inge point remains in an open state. In the ''phosphate'' structure, c losure of the N-terminal hinge point is also evident. Finally and most unexpectedly, both complex structures also contain a 3 Angstrom shift of a helix that lies outside the flexible hinge region. We propose th at a transient shift of this helix is a required element of PGK hinge closure during catalysis. (C) 1998 Academic Press.