Loop‐loop interactions govern multiple steps in indole‐3‐glycerol phosphate synthase catalysis

Abstract

Substrate binding, product release, and likely chemical catalysis in the tryptophan biosynthetic enzyme indole-3-glycerol phosphate synthase (IGPS) are dependent on the structural dynamics of the b1a1 active-site loop. Statistical coupling analysis and molecular dynamic simulations had previously indicated that covarying residues in the b1a1 and b2a2 loops, corresponding to Arg54 and Asn90, respectively, in the Sulfolobus sulfataricus enzyme (ssIGPS), are likely important for coordinating functional motions of these loops. To test this hypothesis, we characterized site mutants at these positions for changes in catalytic function, protein stability and structural dynamics for the thermophilic ssIGPS enzyme. Although there were only modest changes in the overall steady-state kinetic parameters, solvent viscosity and solvent deuterium kinetic isotope effects indicated that these amino acid substitutions change the identity of the rate-determining step across multiple temperatures. Surprisingly, the N90A substitution had a dramatic effect on the general acid/base catalysis of the dehydration step, as indicated by the loss of the descending limb in the pH rate profile, which we had previously assigned to Lys53 on the b1a1 loop. These changes in enzyme function are accompanied with a quenching of ps-ns and ms-ms timescale motions in the b1a1 loop as measured by nuclear magnetic resonance studies. Altogether, our studies provide structural, dynamic and functional rationales for the coevolution of residues on the b1a1 and b2a2 loops, and highlight the multiple roles that the b1a1 loop plays in IGPS catalysis. Thus, substitution of covarying residues in the active-site b1a1 and b2a2 loops of indole-3-glycerol phosphate synthase results in functional, structural, and dynamic changes, highlighting the multiple roles that the b1a1 loop plays in enzyme catalysis and the importance of regulating the structural dynamics of this loop through noncovalent interactions with nearby structural elements.

Controlling Active Site Loop Dynamics in the (β/α)8 Barrel Enzyme Indole-3-Glycerol Phosphate Synthase

“…The R64A/D65A double substitution led to a modest two-fold decrease in the catalytic turnover rate constant (k cat ) and a four-fold decrease in the second-order rate constant (k cat /K M ) at 37 • C compared to WT enzyme (Table 1), similar to the kinetic changes previously reported for the N90A variant [29]. We also performed assays at 75 • C, which is closer to the physiological temperature of S. sulfataricus [31].…”

“…Combined statistical coupling analysis (SCA) and molecular dynamics (MD) simulations had suggested that interactions between the two loops might be important for the function and structure of IGPS [27]. In particular, our experimental studies indicated that the interactions between Arg54 on the β1α1 loop and Asn90 on the β2α2 loop are important for β1α1 loop dynamics and ssIGPS catalysis [29]. For instance, the N90A substitution resulted in a modest decrease in the steady-state kinetic parameters, a different rate-determining step than wild-type (WT) enzyme, and a decrease in the µs-ms timescale motions of the β1α1 loop according to nuclear magnetic resonance (NMR) relaxation studies [29].…”

“…We suggested that there are other interactions that may influence the conformational dynamics of the β1α1 loop [29]. In particular, we noted that there is a hydrogen bond network involving Arg64 and Asp65 on the β1α1 loop, Ile67, Glu68 and Tyr69 on the α1 helix, and Met237 and Arg238 on the α8' helix.…”

“…We have also previously shown that interactions with the nearby β2α2 loop help to govern β1α1 loop dynamics and ssIGPS function [29]. Combined statistical coupling analysis (SCA) and molecular dynamics (MD) simulations had suggested that interactions between the two loops might be important for the function and structure of IGPS [27].…”

“…For instance, the N90A substitution resulted in a modest decrease in the steady-state kinetic parameters, a different rate-determining step than wild-type (WT) enzyme, and a decrease in the µs-ms timescale motions of the β1α1 loop according to nuclear magnetic resonance (NMR) relaxation studies [29]. Surprisingly, severing this interaction between the β1α1 and β2α2 loops also led to an increase in protein stability, as measured by a two-fold decrease in the thermal inactivation rate of the N90A variant compared to the WT enzyme [29].…”