Thermodynamic analysis shows conformational coupling and dynamics confer substrate specificity in fructose-1,6-bisphosphate aldolase

Abstract

Conformational flexibility is emerging as a central theme in enzyme catalysis. Thus, identifying and characterizing enzyme dynamics are critical for understanding catalytic mechanisms. Herein, coupling analysis, which uses thermodynamic analysis to assess cooperativity and coupling between distal regions on an enzyme, is used to interrogate substrate specificity among fructose-1,6-(bis)phosphate aldolase (aldolase) isozymes. Aldolase exists as three isozymes, A, B, and C, distinguished by their unique substrate preferences despite the fact that the structures of the active sites of the three isozymes are nearly identical. While conformational flexibility has been observed in aldolase A, its function in the catalytic reaction of aldolase has not been demonstrated. To explore the role of conformational dynamics in substrate specificity, those residues associated with isozyme specificity (ISRs) were swapped and the resulting chimeras were subjected to steady-state kinetics. Thermodynamic analyses suggest cooperativity between a terminal surface patch (TSP) and a distal surface patch (DSP) of ISRs that are separated by >8.9 A. Notably, the coupling energy (DeltaGI) is anticorrelated with respect to the two substrates, fructose 1,6-bisphosphate and fructose 1-phosphate. The difference in coupling energy with respect to these two substrates accounts for approximately 70% of the energy difference for the ratio of kcat/Km for the two substrates between aldolase A and aldolase B. These nonadditive mutational effects between the TSP and DSP provide functional evidence that coupling interactions arising from conformational flexibility during catalysis are a major determinant of substrate specificity.

Fig 1. The location of aldolase A and B ISRs on the aldolase monomer

A surface model of the aldolase A monomer (pdb code 1ADO) showing the location of aldolase A and B (underlined) ISRs with respect to the active site (two views shown, 180º rotation about y). ISRs of TSP (yellow), the DSP (cyan), and non-patch surface ISRs (green) are labeled and the patches are outlined. Active-site residues are colored red and residue 41, involved in binding the C6-phosphate, is colored gray. The approximate locations of tetramer interfaces are indicated with black bars. The figure was created using PyMOL v0.97 (58).

Fig 2. Diagrammatic representation of aldolase chimeras

Wild-type aldolases A and B, 363 amino acids residues each, are represented as yellow and blue boxes, respectively, above which the ISRs are indicated. The ISRs engineered in each chimera is similarly indicated in color.

Fig 3. Thermodynamic cycles among aldolase chimeras

For each cycle, arrows 1 and 4 indicate the substitution of one set of ISRs, while arrows 2 and 3 indicate the substitution of the other set of ISRs. The diagonal arrow 5 indicates the double substitution. Panel A, ISRs involved in the “Patch” (TSP and DSP) cycle. Panel B, ISRs involved in AB_All (25) (“All”) made with patch (P) and non-patch (NP). Panel C, ISRs involved in the “Non-patch” cycle made with buried (NPB) and surface (NPS).

Fig 4. Ribbon diagram of aldolase depicting a five-helix cluster

The five helices in the cluster involved in determination of substrate specificity are numbered (circled). The ISRs in the TSP (yellow) and the DSP (cyan) are labeled and depicted as sticks. The monomer is orientated with the active-site cleft facing to the right and in the plane of the paper. The distances (Å) between near ISRs in the two patches are indicated in addition to the distance between Cys72 and Cys338 (red sticks). Residues involved in binding the C6-phosphate, Lys41, Arg42, and Arg303 (57) are depicted as gray sticks and the Cα backbone of the CTR is blue. The figure was created using pdb code 1ADO and PyMOL v0.97 (58).

Fig 5. Ribbon diagram of aldolase colored by B-factor

The five helices in the excursion involved in determination of substrate specificity are numbered (circled). The monomer is orientated with the active-site cleft facing to the right and in the plane of the paper. The Cα backbone is colored by increasing B-factor from blue (minimum value is 35 Ų) to red (maximum value is 80 Ų). The figure was created using chain A from pdb code 1ADO and PyMOL v0.97 (58). Other structures in the protein database showed a similar result.

Fig 6. Rates of inactivation by oxidation of Cys72 and Cys338 in the presence of substrates

Aldolases with two reactive surface Cys residues (gBi) was incubated in the presence of cupric phenanthroline and with or without (●) substrates Fru 1,6-P₂ (◇) or Fru 1-P (○). Activity was measured and normalized to the activity at time zero (9.3–12.8 U/mg). Errors were determined for each experiment, which was repeated three times.

Fig 7. Coupling energy associated with k_cat/K_m for both substrates in all three thermodynamic cycles

The ΔG_I (kcal/mol) for k_cat/K_m toward Fru 1,6-P₂ (Panel A) and Fru 1-P (Panel B) were plotted for the indicated thermodynamic cycles. Asterisks (*) indicate significant non-additivity of ΔG_I values different from zero by student t-test (p < 0.001).