Recent evidence shows dynamic fluctuations of structure are essential components of enzyme catalysis (1
). While it is likely that most enzymes exhibit flexibility as part of the catalytic process, the role of this movement is enzyme specific. For example, in the case of dihydrofolate reductase (DHFR), backbone and side-chain motions are essential for cofactor binding, substrate binding, and catalysis (2
). Thus, identifying and characterizing these motions is paramount to understanding enzyme mechanisms.
Recently, the importance of conformational flexibility has been highlighted in the study of ligand binding and catalysis, as inferred from X-ray crystallographic and NMR structures as well as computational analysis. The term “allostery” has been applied to the transmission of energy resulting from structural rearrangement and/or dynamic changes within a single-domain protein (3
). In addition to structural and computational data, evolutionary data for a protein family has been used to show that conserved sets of interacting residues form connected pathways through the protein to transmit changes upon ligand binding (6
). Not only ligand binding but catalysis is dependent upon inherent mobility of a protein during the catalytic cycle with one or more of the catalytic steps being reflected in an intrinsic motion of the enzyme (for review see (1
) and for a recent example (7
)). Thus, the innate mobility of the protein scaffold is manifested in the various steps of the catalytic cycle. Indeed, extensive studies of hydrogen transfer in proteins are consistent with the concept that protein dynamics create transient heavy-atom configurations with a favorable electrostatic environment for proton and hydride transfer (1
). In comparison, determining how conformational flexibility affords the remarkable selectivity of enzymes has received less effort. However, recent work on T7-DNA polymerase has invoked a role for enzyme dynamics in distinguishing correct nucleotide incorporation from misincorporation (13
Here, multiple mutational analyses in fructose-1,6-(bis)phosphate aldolase (aldolase, EC 220.127.116.11) are used to identify and characterize conformational coupling as a potential mechanism conferring substrate specificity among aldolase isozymes. Aldolase catalyzes the reversible C-C bond cleavage of fructose 1,6-(bis)phosphate (Fru 1,6-P2
) and fructose 1-phosphate (Fru 1-P) in its roles in glycolysis, gluconeogenesis, and fructose metabolism (14
). Aldolase isozymes are distinguished by their unique expression profiles and substrate specificities (15
). Aldolase A is expressed primarily in skeletal muscle and has the highest turnover toward Fru 1,6-P2
of the three isozymes (15
). Aldolase B is expressed primarily in liver and kidney where most Fru-1-P degradation takes place (14
). While the catalytic mechanism of aldolase is well characterized (19
), and crystal structures of all three human isozymes have been determined (22
), it is still unclear how aldolase catalyzes the cleavage of the two substrates with distinct and physiologically relevant efficiencies. For example, consistent with its role in gluconeogenesis, aldolase B has equal turnover numbers for the two hexose substrates and has a lower Km
value for glyceraldehyde 3-phosphate and dihydroxyacetone phosphate than aldolase A (17
). Correspondingly, aldolase A has evolved to cleave Fru 1,6-P2
more efficiently than Fru 1-P (as evidenced by the kcat
ratio for Fru 1,6-P2
:Fru 1-P of 70,000). This ratio for aldolase B is ~900, therefore the evolution of these parameters for aldolase A compared to that of aldolase B has resulted in a ~80-fold (70,000/900) difference in selectivity between the two hexoses.
Previous work attempting to decipher the root of isozyme specificity has identified a set of residues conserved among, but not between each isozyme, termed isozyme-specific residues (ISRs). Surprisingly, ISRs are not located at or near the active site, but mostly (24 out of 27) fall on the surface of the protein (25
). Moreover, a large proportion of ISRs are found in the C-terminal region (CTR) of the enzyme. The role of ISRs was previously examined by swapping ISRs from aldolase B into aldolase A demonstrating that the ISRs are necessary and sufficient to confer kinetic parameters (kcat
) of aldolase B onto aldolase A (25
). Because of their conservation and involvement in conferring substrate specificity, it is hypothesized that interactions between ISRs are responsible for altering the structure of the active site from a distance.
One means by which enzymes may confer substrate specificity at a distance would involve dynamic cooperation/coupling among sets of ISRs. Herein, multiple mutational analyses were employed as a novel method to attribute the role of conformational coupling to substrate specificity among aldolase isozymes. Traditionally, this analysis is performed by making two separate, single-residue mutations and the corresponding double mutation, calculating the change in free energy (ΔΔG) associated with these changes, and comparing the change in free energy with that of the double mutant (9
). If the changes in free energy of the two single mutants do not sum to the change in free energy for the double mutant, then a cooperative/coupling mechanism between the residues is invoked. To perform the analogous experiment for aldolase ISRs, groups of mutations were analyzed rather than single amino-acid substitutions. Multiple mutational analyses revealed that substrate specificity of aldolase A was conferred by cooperative effects between two distantly located surface patches, one of which includes the CTR. This analysis exposes a means by which these cooperative effects among ISRs affect substrate specificity in enzymes with seemingly identical active sites.