Biophysical studies of the dynamics and

mechanism of Class II fructose 1,6-bisphosphate aldolase

Christine Hilcenko, Arnout P. Kalverda, Steve W. Homans, Alan Berry.

 

Introduction

The importance of protein dynamics in enzyme catalysis and specificity has been previously highlighted in a number of proteins, such as hexokinase and carboxypeptidase A. We know that motions in proteins, ranging from fluctuation of atoms or side chains to closure of entire loops, are a prerequisite for substrates to bind and for reaction products to leave. For example, the role of such flexible loops has been studied in the archetypal (a /b )8-barrel protein, triose phosphate isomerase, where a loop, between residues 166-176, in the protein moves from an open position, when substrate is not bound, to a closed position when the substrate binds, stabilizing the reaction intermediate.

Another important member of the (a /b )8-barrel family is fructose-1,6-bisphosphate aldolase (FBP-aldolase), which catalyses the reversible aldol condensation of glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) to form FBP. The FBP-aldolases can be divided into Class I and II enzymes, where the latter is homodimeric and requires one zinc ion per monomer for catalysis.

Motions in the mechanism of Class II FBP-aldolase

The availability of several crystal structures of the Class II FBP-aldolase has revealed differences in zinc binding in each distinct structure. Comparison of the structures in the absence or presence of the substrate analogue, phosphoglycolohydroxamate, suggests that rotation of His-110 and His-264 upon substrate binding moves the zinc from a buried site to a more exposed one where it binds to the substrate. Other motions in the enzyme on substrate binding include the movement of the loop containing the highly conserved Glu-182, the catalytic base responsible for proton abstraction from DHAP.

Multi-dimensional NMR spectroscopy

A programme of high-field NMR spectroscopy has been initiated to study the movement of this flexible loop and the role of motion in enzyme catalysis and specificity. The challenging aspect of this project is to work with such a large molecule, a dimer of 78kDa. A number of labelled samples have been prepared containing 2H, 15N and 13C. Over-expression studies show that high levels of FBP-aldolase over-expression, similar to that obtained in rich media, can be achieved in the labelling media with D2O. Both [1H, 15N]-HSQC and [1H, 15N]-TROSY spectra have been collected and we are now using TROSY type triple-resonance NMR experiments to determine the backbone chemical shifts of the residues of interest in the active site and in the flexible loop.

 

 

SDS-PAGE analysis of the various stages of the purification of [15N, 13C, 2H]-FBP-aldolase.

Lane 1: Molecular weight markers.
Lane 2: Whole cell extract.
Lane 3: After the French Press.
Lane 4: 40-80% ammonium sulphate precipitate.
Lane 5: DE-52 anion exchange eluate.

Portion of the 600 MHz [1H, 15N]-TROSY spectrum of [15N, 13C, 2H]-FBP-aldolase.

References

Zgiby, S. M., Thomson, G.J., Qamar, S. & Berry, A. (2000) Exploring substrate binding and discrimination in fructose-1,6-bisphosphate and tagatose-1,6-bisphosphate aldolases. Eur. J. Biochem, 267, 1858-1868.

Hall, D. R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) The crystal structure of Escherichia coli Class II fructose-1,6-bisphosphate aldolase in complex with phosphoglycolohydroxamate reveals details of mechanism and specificity. J. Mol. Biol., 287, 383-394.

Plater, A. R., Zgiby, S.M., Thomson, G.J., Qamar, S., Wharton, C.W. & Berry, A. (1999) Conserved residues in the mechanism of the E. coli Class II FBP-aldolase. J. Mol. Biol., 285, 843-855.

Funding: BBSRC and The Wellcome Trust are gratefully acknowledged.