STRUCTURAL INVESTIGATION OF REGULATION IN BACILLUS STEAROTHERMOPHILUS PYRUVATE KINASE. Simon C Lovell1, Daniel Ungar2, Abdul H Mullick2 and Hilary Muirhead2. 1Biochemistry Dept, University of Bath, Claverton Down, Bath; 2Biochemistry Dept, University of Bristol, University Walk, Bristol
A variety of techniques are being used to study enzyme regulation using the glycolytic enzyme pyruvate kinase, using the moderate thermophile Bacillus stearothermophilus as a model system. Our approach involves the use of X-ray crystallography, site directed mutaganesis, fluorescence and both steady state and transient kinetics.
Pyruvate kinase is a tetrameric enzyme and catalyses the reaction:
PEP + Mg.ADP ------------> Pyruvate + Mg.ATP + H+
We have demonstrated the existence of at least four distinct conformations for B stearothermophilus pyruvate kinase, which is activated by ribose-5-phosphate (1). These structures correspond to the unliganded, phosphoenolpyruvate (PEP) bound, activator bound and fully liganded species. Structures have been published for the unliganded enzyme from E coli (2) and the constitutively activated forms from muscle (3,4). In order to understand the regulation of this complex enzyme it will be necessary to know the structure of all of the above conformations - preferably from the same species.
We have determined the structure of the unliganded of the B stearothermophilus enzyme at a resolution of 3.2 Å. The evidence for this enzyme having a T-state like conformation is presented and the differences between it and the E coli structure are described. B stearothermophilus pyruvate kinase has a 113 amino acid C-terminal extension when compared to all other pyruvate kinases of known sequence (5). Surprisingly the removal of this extension has very little effect on either activity or regulation. At the current resolution and state of refinement this extension cannot be seen in the crystal structure, although a cavity of the appropriate size and position exists.
1 Lovell, Mullick and Muirhead, in preparation.
2 Mattevi et al (1995) Structure 3 729
3 Muirhead et al (1986) EMBO J 5 475
4 Larsen et al (1994) Biochemistry 33 6301
5 Sakai and Ohta (1993) Eur J Biochem 211 851