Pre-Steady-State Kinetic Analysis of 1-Deoxy-d-xylulose-5-phosphate Reductoisomerase from <i>Mycobacterium tuberculosis</i> Reveals Partially Rate-Limiting Product Release by Parallel Pathways

As part of the non-mevalonate pathway for the biosynthesis of the isoprenoid precursor isopentenyl pyrophosphate, 1-deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase (DXR) catalyzes the conversion of DXP into 2-<i>C</i>-methyl-d-erythritol 4-phosphate (MEP) by consecutive isomerization and NADPH-dependent reduction reactions. Because this pathway is essential to many infectious organisms but is absent in humans, DXR is a target for drug discovery. In an attempt to characterize its kinetic mechanism and identify rate-limiting steps, we present the first complete transient kinetic investigation of DXR. Stopped-flow fluorescence measurements with <i>Mycobacterium tuberculosis</i> DXR (<i>Mt</i>DXR) revealed that NADPH and MEP bind to the free enzyme and that the two bind together to generate a nonproductive ternary complex. Unlike the <i>Escherichia coli</i> orthologue, <i>Mt</i>DXR exhibited a burst in the oxidation of NADPH during pre-steady-state reactions, indicating a partially rate-limiting step follows chemistry. By monitoring NADPH fluorescence during these experiments, the transient generation of <i>Mt</i>DXR·NADPH·MEP was observed. Global kinetic analysis supports a model involving random substrate binding and ordered release of NADP⁺ followed by MEP. The partially rate-limiting release of MEP occurs via two pathwaysdirectly from the binary complex and indirectly via the <i>Mt</i>DXR·NADPH·MEP complexthe partitioning being dependent on NADPH concentration. Previous mechanistic studies, including kinetic isotope effects and product inhibition, are discussed in light of this kinetic mechanism

DXP Reductoisomerase: Reaction of the Substrate in Pieces Reveals a Catalytic Role for the Nonreacting Phosphodianion Group

The role of the nonreacting phosphodianion group of 1-deoxy-d-xylulose-5-phosphate (DXP) in catalysis by DXP reductoisomerase (DXR) was investigated for the reaction of the “substrate in pieces”. The truncated substrate 1-deoxy-l-erythrulose is converted by DXR to 2-<i>C</i>-methylglycerol with a <i>k</i>_cat/<i>K</i>_m that is 10⁶-fold lower than that for DXP. Phosphite dianion was found to be a nonessential activator, providing 3.2 kcal/mol of transition state stabilization for the truncated substrate. These results implicate a phosphate-driven conformational change involving loop closure over the DXR active site to generate an environment poised for catalysis

Cysteine Is the General Base That Serves in Catalysis by Isocitrate Lyase and in Mechanism-Based Inhibition by 3‑Nitropropionate

Isocitrate lyase (ICL) catalyzes the reversible cleavage of isocitrate into succinate and glyoxylate. It is the first committed step in the glyoxylate cycle used by some organisms, including <i>Mycobacterium tuberculosis</i>, where it has been shown to be essential for cell survival during chronic infection. The pH–rate and pD–rate profiles measured in the direction of isocitrate synthesis revealed solvent kinetic isotope effects (KIEs) of 1.7 ± 0.4 for ^D₂O<i>V</i> and 0.56 ± 0.07 for ^D₂O(<i>V</i>/<i>K</i>_succinate). Whereas the ^D₂O<i>V</i> is consistent with partially rate-limiting proton transfer during formation of the hydroxyl group of isocitrate, the large inverse ^D₂O(<i>V</i>/<i>K</i>_succinate) indicates that substantially different kinetic parameters exist when the enzyme is saturated with succinate. Inhibition by 3-nitropropionate (3-NP), a succinate analogue, was found to proceed through an unusual double slow-onset process featuring formation of a complex with a <i>K</i>_i of 3.3 ± 0.2 μM during the first minute, followed by formation of a final complex with a <i>K</i>_i* of 44 ± 10 nM over the course of several minutes to hours. Stopped-flow measurements during the first minute revealed an apparent solvent KIE of 0.40 ± 0.03 for association and unity for dissociation. In contrast, itaconate, a succinate analogue lacking an acidic α-proton, did not display slow-binding behavior and yielded a ^D₂O<i>K</i>_i of 1.0 ± 0.2. These results support a common mechanism for catalysis with succinate and inhibition by 3-NP featuring (1) an unfavorable prebinding isomerization of the active site Cys191–His193 pair to the thiolate–imidazolium form, a process that is favored in D₂O, and (2) the transfer of a proton from succinate or 3-NP to Cys191. These findings also indicate that propionate-3-nitronate, which is the conjugate base of 3-NP and the “true inhibitor” of ICL, does not bind directly and must be generated enzymatically

The Nitro Group as a Masked Electrophile in Covalent Enzyme Inhibition

We report the unprecedented reaction between a nitroalkane and an active-site cysteine residue to yield a thiohydroximate adduct. Structural and kinetic evidence suggests the nitro group is activated by conversion to its nitronic acid tautomer within the active site. The nitro group, therefore, shows promise as a masked electrophile in the design of covalent inhibitors targeting binding pockets with appropriately placed cysteine and general acid residues

The Role of Phosphate in a Multistep Enzymatic Reaction: Reactions of the Substrate and Intermediate in Pieces

Several mechanistically unrelated enzymes utilize the binding energy of their substrate’s nonreacting phosphoryl group to accelerate catalysis. Evidence for the involvement of the phosphodianion in transition state formation has come from reactions of the substrate in pieces, in which reaction of a truncated substrate lacking its phosphorylmethyl group is activated by inorganic phosphite. What has remained unknown until now is how the phosphodianion group influences the reaction energetics at different points along the reaction coordinate. 1-Deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase (DXR), which catalyzes the isomerization of DXP to 2-<i>C</i>-methyl-d-erythrose 4-phosphate (MEsP) and subsequent NADPH-dependent reduction, presents a unique opportunity to address this concern. Previously, we have reported the effect of covalently linked phosphate on the energetics of DXP turnover. Through the use of chemically synthesized MEsP and its phosphate-truncated analogue, 2-<i>C</i>-methyl-d-glyceraldehyde, the current study revealed a loss of 6.1 kcal/mol of kinetic barrier stabilization upon truncation, of which 4.4 kcal/mol was regained in the presence of phosphite dianion. The activating effect of phosphite was accompanied by apparent tightening of its interactions within the active site at the intermediate stage of the reaction, suggesting a role of the phosphodianion in disfavoring intermediate release and in modulation of the on-enzyme isomerization equilibrium. The results of kinetic isotope effect and structural studies indicate rate limitation by physical steps when the covalent linkage is severed. These striking differences in the energetics of the natural reaction and the reactions in pieces provide a deeper insight into the contribution of enzyme–phosphodianion interactions to the reaction coordinate

Highly Precise Measurement of Kinetic Isotope Effects Using ¹H‑Detected 2D [¹³C,¹H]-HSQC NMR Spectroscopy

A new method is presented for measuring kinetic isotope effects (KIEs) by ¹H-detected 2D [¹³C,¹H]-heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The high accuracy of this approach was exemplified for the reaction catalyzed by glucose-6-phosphate dehydrogenase by comparing the 1-¹³C KIE with the published value obtained using isotope ratio mass spectrometry. High precision was demonstrated for the reaction catalyzed by 1-deoxy-d-xylulose-5-phosphate reductoisomerase from Mycobacterium tuberculosis. 2-, 3-, and 4-¹³C KIEs were found to be 1.0031(4), 1.0303(12), and 1.0148(2), respectively. These KIEs provide evidence for a cleanly rate-limiting retroaldol step during isomerization. The high intrinsic sensitivity and signal dispersion of 2D [¹³C,¹H]-HSQC offer new avenues to study challenging systems where low substrate concentration and/or signal overlap impedes 1D ¹³C NMR data acquisition. Moreover, this approach can take advantage of highest-field spectrometers, which are commonly equipped for ¹H detection with cryogenic probes

Alteration of the Flexible Loop in 1‑Deoxy‑d‑xylulose-5-phosphate Reductoisomerase Boosts Enthalpy-Driven Inhibition by Fosmidomycin

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), which catalyzes the first committed step in the 2-<i>C</i>-methyl-d-erythritol 4-phosphate pathway of isoprenoid biosynthesis used by <i>Mycobacterium tuberculosis</i> and other infectious microorganisms, is absent in humans and therefore an attractive drug target. Fosmidomycin is a nanomolar inhibitor of DXR, but despite great efforts, few analogues with comparable potency have been developed. DXR contains a strictly conserved residue, Trp203, within a flexible loop that closes over and interacts with the bound inhibitor. We report that while mutation to Ala or Gly abolishes activity, mutation to Phe and Tyr only modestly impacts <i>k</i>_cat and <i>K</i>_m. Moreover, pre-steady-state kinetics and primary deuterium kinetic isotope effects indicate that while turnover is largely limited by product release for the wild-type enzyme, chemistry is significantly more rate-limiting for W203F and W203Y. Surprisingly, these mutants are more sensitive to inhibition by fosmidomycin, resulting in <i>K</i>_m/<i>K</i>_i ratios up to 19-fold higher than that of wild-type DXR. In agreement, isothermal titration calorimetry revealed that fosmidomycin binds up to 11-fold more tightly to these mutants. Most strikingly, mutation strongly tips the entropy–enthalpy balance of total binding energy from 50% to 75% and 91% enthalpy in W203F and W203Y, respectively. X-ray crystal structures suggest that these enthalpy differences may be linked to differences in hydrogen bond interactions involving a water network connecting fosmidomycin’s phosphonate group to the protein. These results confirm the importance of the flexible loop, in particular Trp203, in ligand binding and suggest that improved inhibitor affinity may be obtained against the wild-type protein by introducing interactions with this loop and/or the surrounding structured water network