Exploring substrate binding and discrimination in fructose1,6-bisphosphateand tagatose 1,6-bisphosphate aldolases

Fructose 1,6-bisphosphate aldolase catalyses the reversible condensation of glycerone-P and glyceraldehyde 3-phosphate into fructose 1,6-bisphosphate. A recent structure of the Escherichia coli Class II fructose 1,6-bisphosph ate aldolase [Hall, D.R., Leonard, G.A., Reed, C.D., Watt, C.I., Berry, A. & Hunter, W.N. (1999) J. Mol. Biol. 287, 383-394] in the presence of the tr ansition state analogue phosphoglycolohydroxamate delineated the roles of i ndividual amino acids in binding glycerone-P and in the initial proton abst raction steps of the mechanism. The X-ray structure has now been used, toge ther with sequence alignments, site-directed mutagenesis and steady-state e nzyme kinetics to extend these studies to map important residues in the bin ding of glyceraldehyde 3-phosphate. From these studies three residues (Asn3 5, Ser61 and Lys325) have been identified as important in catalysis. We sho w that mutation of Ser61 to alanine increases the K-m value for fructose 1, 6-bisphosphate 16-fold and product inhibition studies indicate that this ef fect is manifested most strongly in the glyceraldehyde 3-phosphate binding pocket of the active site, demonstrating that Ser61 is involved in binding glyceraldehyde 3-phosphate. In contrast a S61T mutant had no effect on cata lysis emphasizing the importance of an hydroxyl group for this role. Mutati on of Asn35 (N35A) resulted in an enzyme with only 1.5% of the activity of the wild-type enzyme and different partial reactions indicate that this res idue effects the binding of both triose substrates. Finally, mutation of Ly s325 has a greater effect on catalysis than on binding, however, given the magnitude of the effects it is likely that it plays an indirect role in mai ntaining other critical residues in a catalytically competent conformation. Interestingly, despite its proximity to the active site and high sequence conservation, replacement of a fourth residue, Gln59 (Q59A) had no signific ant effect on the function of the enzyme. In a separate study to characteri ze the molecular basis of aldolase specificity, the agaY-encoded tagatose 1 ,6-bisphosphate aldolase of E. coli was cloned, expressed and kinetically c haracterized. Our studies showed that the two aldolases are highly discrimi nating between the diastereoisomers fructose bisphosphate and tagatose bisp hosphate, each enzyme preferring its cognate substrate by a factor of 300-1 500-fold. This produces an overall discrimination factor of almost 5 x 10(5 ) between the two enzymes. Using the X-ray structure of the fructose 1,6-bi sphosphate aldolase and multiple sequence alignments, several residues were identified, which are highly conserved and are in the vicinity of the acti ve site. These residues might potentially be important in substrate recogni tion. As a consequence, nine mutations were made in attempts to switch the specificity of the fructose 1,6-bisphosphate aldolase to that of the tagato se 1,6-bisphosphate aldolase and the effect on substrate discrimination was evaluated. Surprisingly, despite making multiple changes in the active sit e, many of which abolished fructose 1,6-bisphosphate aldolase activity, no switch in specificity was observed. This highlights the complexity of enzym e catalysis in this family of enzymes, and points to the need for further s tructural studies before we fully understand the subtleties of the shaping of the active site for complementarity to the cognate substrate.