Slowed Enzymatic Turnover Allows Characterization of Intermediates by Solid-State NMR † (original) (raw)
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Structure of Carbamoyl Phosphate Synthetase: A Journey of 96 Å from Substrate to Product
Biochemistry, 1997
Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. As isolated from Escherichia coli, the enzyme has a total molecular weight of ∼160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal structure of this enzyme determined to 2.8 Å resolution in the presence of ADP, Mn 2+ , phosphate, and ornithine. The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH 2-and COOH-terminal domains. Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components. Each component contains four distinct domains. Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase. A linear distance of ∼80 Å separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit. The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein. † Supported in part by NIH Grants DK47814 (H.M.H.), DK30343 (F.M.R.), and AR35186 (I.R.) and the Robert A. Welch Foundation (A-840) (F.M.R.). ‡ Coordinates have been deposited in the Brookhaven Protein Data Bank (1JDB).
Structure of Carbamoyl Phosphate Synthetase: A Journey of 96 Å from Substrate to Product,
Biochemistry, 1997
Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. As isolated from Escherichia coli, the enzyme has a total molecular weight of ∼160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal structure of this enzyme determined to 2.8 Å resolution in the presence of ADP, Mn 2+ , phosphate, and ornithine. The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH 2-and COOH-terminal domains. Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components. Each component contains four distinct domains. Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase. A linear distance of ∼80 Å separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit. The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein. † Supported in part by NIH Grants DK47814 (H.M.H.), DK30343 (F.M.R.), and AR35186 (I.R.) and the Robert A. Welch Foundation (A-840) (F.M.R.). ‡ Coordinates have been deposited in the Brookhaven Protein Data Bank (1JDB).
Biochemical and Biophysical Research Communications, 1988
In order to detect covalent reaction intermediates in the 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase reaction, we have investigated the interaction of EPSP synthase with the reaction product EPSP. An exchange of EPSP-methylene protons could be demonstrated by incubating EPSPS with EPSP in D20. Since trace amounts of contaminating Pi would lead to reversal of EPSPS reaction and hence methylene proton exchange, we added pyruvate kinase, ADP, Mg++ and K+. Under these conditions, any contaminating Pi that is converted to PEP is trapped as ATP. No exchange of EPSP protons with those of the solvent could be detected in the presence of this trap system, suggesting that enzyme-bound EPSP is unable to form a covalent tetrahedral complex. Incorporation of [14C] from [14C]-$3P and [14C]-PEP into EPSP could be detected, but only in the absence of a PEP (or Pi) trap system. This indicates that for the exchange reaction, Pi is required, and also indicates the absence of a covalent intermediate, unless the carboxyvinyl-enzyme-bound S3P is completely restricted from exchange. ® 1988 Academic Press, Inc. 5-Enolpyruvylshikimate-3-phosphate synthase (EPSP2 synthase, EPSPS) (EC 2.5.1.19) catalyzes the formation of EPSP from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP) in both plants and microorganisms. EPSP is a precursor of chorismate, an intermediate of the shikimate pathway involved in the biosynthesis of a number of aromatic compounds such as aromatic amino acids and vitamins . There is significant interest in EPSPS since it is a target for the herbicide glyphosate.
Journal of Molecular Biology, 1996
The 46 kDa enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase Department of Chemistry Washington University catalyzes the condensation of shikimate-3-phosphate (S3P) and phospho-St. Louis, MO 63130, USA enolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. As part of a solid-state NMR characterization of this structure, 15 N labels were introduced selectively into the lysine, arginine and histidine residues of EPSP synthase and distances to a 13 C label in Glp and to the 31 P in S3P and Glp were measured by rotational-echo double-resonance NMR. Three lysine and four arginine residues are in the proximity of the phosphate group of S3P and the carboxyl and phosphonate groups of Glp. A single histidine residue is in the vicinity of the binding site (closer to Glp than to S3P) but is more distant than the lysine and arginine residues.
Observation of Covalent Intermediates in an Enzyme Mechanism at Atomic Resolution
Science, 2001
In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra–high resolution structures of wild-type and mutant d -2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and 1 H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.
Journal of Biomolecular NMR, 2000
The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase-S3P-Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native 31 P in S3P and Glp, biosynthetically 13 C-labeled S3P, specifically 13 C and 15 N labeled Glp, and a variety of protein-15 N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.
The Small Subunit of Carbamoyl Phosphate Synthetase: Snapshots along the Reaction Pathway
Biochemistry, 1999
Carbamoyl phosphate synthetase (CPS) plays a key role in both arginine and pyrimidine biosynthesis by catalyzing the production of carbamoyl phosphate. The enzyme from Escherichi coli consists of two polypeptide chains referred to as the small and large subunits. On the basis of both amino acid sequence analyses and X-ray structural studies, it is known that the small subunit belongs to the Triad or Type I class of amidotransferases, all of which contain a cysteine-histidine (Cys269 and His353) couple required for activity. The hydrolysis of glutamine by the small subunit has been proposed to occur via two tetrahedral intermediates and a glutamyl-thioester moiety. Here, we describe the three-dimensional structures of the C269S/glutamine and CPS/glutamate γ-semialdehyde complexes, which serve as mimics for the Michaelis complex and the tetrahedral intermediates, respectively. In conjunction with the previously solved glutamyl-thioester intermediate complex, the stereochemical course of glutamine hydrolysis in CPS has been outlined. Specifically, attack by the thiolate of Cys269 occurs at the Si face of the carboxamide group of the glutamine substrate leading to a tetrahedral intermediate with an S-configuration. Both the backbone amide groups of Gly241 and Leu270, and O γ of Ser47 play key roles in stabilizing the developing oxyanion. Collapse of the tetrahedral intermediate leads to formation of the glutamyl-thioester intermediate, which is subsequently attacked at the Si face by an activated water molecule positioned near His353. The results described here serve as a paradigm for other members of the Triad class of amidotranferases.
Proceedings of the National Academy of Sciences, 2001
Recent developments in NMR have extended the size range of proteins amenable to structural and functional characterization to include many larger proteins involved in important cellular processes. By applying a combination of residue-specific isotope labeling and protein deuteration strategies tailored to yield specific information, we were able to determine the solution structure and study structure-activity relationships of 3,4-dihydroxy-2-butanone-4-phosphate synthase, a 47-kDa enzyme from the Escherichia coli riboflavin biosynthesis pathway and an attractive target for novel antibiotics. Our investigations of the enzyme's ligand binding by NMR and site-directed mutagenesis yields a conclusive picture of the location and identity of residues directly involved in substrate binding and catalysis. Our studies illustrate the power of state-of-the-art NMR techniques for the structural characterization and investigation of ligand binding in protein complexes approaching the 50-kDa range in solution.
Biochemistry, 2001
The crystal structures of 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDOPS) from Escherichia coli complexed with the substrate phosphoenolpyruvate (PEP) and with a mechanism-based inhibitor (K d ) 0.4 µM) were determined by molecular replacement using X-ray diffraction data to 2.8 and 2.3 Å resolution, respectively. Both the KDOPS‚PEP and KDOPS‚inhibitor complexes crystallize in the cubic space group I23 with cell constants a ) b ) c ) 117.9 and 117.6 Å, respectively, and one subunit per asymmetric unit. The two structures are nearly identical, and superposition of their CR atoms indicates an rms difference of 0.41 Å. The PEP in the KDOPS‚PEP complex is anchored to the enzyme in a conformation that blocks its si face and leaves its re face largely devoid of contacts. This results from KDOPS's selective choice of a PEP conformer in which the phosphate group of PEP is extended toward the si face. Furthermore, the structure reveals that the bridging (P-O-C) oxygen atom and the carboxylate group of PEP are not strongly hydrogen-bonded to the enzyme. The resulting high degree of negative charge on the carboxylate group of PEP would then suggest that the condensation step between PEP and D-arabinose-5-phosphate (A5P) should proceed in a stepwise fashion through the intermediacy of a transient oxocarbenium ion at C2 of PEP. The molecular structural results are discussed in light of the chemically similar but mechanistically distinct reaction that is catalyzed by the enzyme 3-deoxy-Darabino-2-heptulosonate-7-phosphate synthase and in light of the preferred enzyme-bound states of the substrate A5P.