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Title: Observation by sup 13 C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site

Abstract

Direct observation of the tetrahedral intermediate in the EPSP synthase reaction pathway was provided by {sup 13}C NMR by examining the species bound to the enzyme active site under internal equilibrium conditions and using (2-{sup 13}C)PEP as a spectroscopic probe. The tetrahedral center of the intermediate bound to the enzyme gave a unique signal appearing at 104 ppm. Separate signals were observed for free EPSP and EPSP bound to the enzyme in a ternary complex with phosphate. These peak assignments account for the quantitation of the species bound to the enzyme and liberated upon quenching with either triethylamine or base. A comparison of quenching with acid, base, or triethylamine was conducted. After long times of incubation during the NMR measurement, a signal at 107 ppm appeared. The compound giving rise to this resonance was isolated and identified as an EPSP ketal. The rate of formation of the EPSP ketal was very slow establishing that it is a side product of the normal enzymatic reaction. To look for additional signals that might arise from a covalent adduct which has been postulated to arise from reaction of enzyme with PEP, and NMR experiment was performed with an analogue of S3P lacking themore » 4- and 5-hydroxyl groups. All of these results reaffirm identification of the tetrahedral species as the only observable intermediate in the EPSP synthase reaction.« less

Authors:
; ; ;  [1]; ;  [2]
  1. (Monsanto Agricultural Company, St. Louis, MO (USA))
  2. (Pennsylvania State Univ., University Park (USA))
Publication Date:
OSTI Identifier:
7100660
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (USA); Journal Volume: 29:6
Country of Publication:
United States
Language:
English
Subject:
62 RADIOLOGY AND NUCLEAR MEDICINE; LIGASES; NUCLEAR MAGNETIC RESONANCE; SHIKIMIC ACID; BIOLOGICAL PATHWAYS; CARBON 13; LIQUID COLUMN CHROMATOGRAPHY; NMR SPECTRA; PHOSPHOENOLPYRUVATE; CARBON ISOTOPES; CARBOXYLIC ACIDS; CHROMATOGRAPHY; ENZYMES; EVEN-ODD NUCLEI; HYDROXY ACIDS; ISOTOPES; LIGHT NUCLEI; MAGNETIC RESONANCE; NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; RESONANCE; SEPARATION PROCESSES; SPECTRA; STABLE ISOTOPES; 550601* - Medicine- Unsealed Radionuclides in Diagnostics

Citation Formats

Anderson, K.S., Sammons, R.D., Leo, G.C., Sikorski, J.A., Benesi, A.J., and Johnson, K.A. Observation by sup 13 C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site. United States: N. p., 1990. Web. doi:10.1021/bi00458a017.
Anderson, K.S., Sammons, R.D., Leo, G.C., Sikorski, J.A., Benesi, A.J., & Johnson, K.A. Observation by sup 13 C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site. United States. doi:10.1021/bi00458a017.
Anderson, K.S., Sammons, R.D., Leo, G.C., Sikorski, J.A., Benesi, A.J., and Johnson, K.A. Tue . "Observation by sup 13 C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site". United States. doi:10.1021/bi00458a017.
@article{osti_7100660,
title = {Observation by sup 13 C NMR of the EPSP synthase tetrahedral intermediate bound to the enzyme active site},
author = {Anderson, K.S. and Sammons, R.D. and Leo, G.C. and Sikorski, J.A. and Benesi, A.J. and Johnson, K.A.},
abstractNote = {Direct observation of the tetrahedral intermediate in the EPSP synthase reaction pathway was provided by {sup 13}C NMR by examining the species bound to the enzyme active site under internal equilibrium conditions and using (2-{sup 13}C)PEP as a spectroscopic probe. The tetrahedral center of the intermediate bound to the enzyme gave a unique signal appearing at 104 ppm. Separate signals were observed for free EPSP and EPSP bound to the enzyme in a ternary complex with phosphate. These peak assignments account for the quantitation of the species bound to the enzyme and liberated upon quenching with either triethylamine or base. A comparison of quenching with acid, base, or triethylamine was conducted. After long times of incubation during the NMR measurement, a signal at 107 ppm appeared. The compound giving rise to this resonance was isolated and identified as an EPSP ketal. The rate of formation of the EPSP ketal was very slow establishing that it is a side product of the normal enzymatic reaction. To look for additional signals that might arise from a covalent adduct which has been postulated to arise from reaction of enzyme with PEP, and NMR experiment was performed with an analogue of S3P lacking the 4- and 5-hydroxyl groups. All of these results reaffirm identification of the tetrahedral species as the only observable intermediate in the EPSP synthase reaction.},
doi = {10.1021/bi00458a017},
journal = {Biochemistry; (USA)},
number = ,
volume = 29:6,
place = {United States},
year = {Tue Feb 13 00:00:00 EST 1990},
month = {Tue Feb 13 00:00:00 EST 1990}
}
  • Porphobilinogen synthase (PBGS) catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). Despite the 280,000-dalton size of PBGS, much can be learned about the reaction mechanism through {sup 13}C and {sup 15}N NMR. The authors knowledge, these studies represent the largest protein complex for which individual nuclei have been characterized by {sup 13}C or {sup 15}N NMR. Here they extend their {sup 13}C NMR studies to PBGS complexes with (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA and report {sup 15}N NMR studies of ({sup 15}N)ALA bound to PBGS. As in their previous {sup 13}C NMR studies, observation of enzyme-bound {sup 15}N-labeledmore » species was facilitated by deuteration at nitrogens that are attached to slowly exchanging hydrogens. For holo-PBGS at neutral pH, the NMR spectra reflect the structure of the enzyme-bound product porphobilinogen (PBG), whose chemical shifts are uniformly consistent with deprotonation of the amino group whose solution pK{sub a} is 11. Despite this local environment, the protons of the amino group are in rapid exchange with solvent. For methyl methanethiosulfonate (MMTS) modified PBGS, the NMR spectra reflect the chemistry of an enzyme-bound Schiff base intermediate that is formed between C{sub 4} of ALA and an active-site lysine. The {sup 13}C chemical shift of (3,3-{sup 2}H{sub 2},3-{sup 13}C)ALA confirms that the Schiff base is an imine of E stereochemistry. By comparison to model imines formed between ({sup 15}N)ALA and hydrazine or hydroxylamine, the {sup 15}N chemical shift of the enzyme-bound Schiff base suggests that the free amino group is an environment resembling partial deprotonation. Deprotonation of the amino group would facilitate formation of a Schiff base between the amino group of the enzyme-bound Schiff base and C{sub 4} of the second ALA substrate. This is the first evidence supporting carbon-nitrogen bond formation as the initial site of interaction between the two substrate molecules.« less
  • /sup 13/C NMR has been used to observe the equilibrium complex of (4-/sup 13/C)-5-aminolevulinate ((4-/sup 13/C)ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. (4-/sup 13/C)ALA (chemical shift = 205.9 ppm) forms (3,5-/sup 13/C)PBG (chemical shifts = 121.0 and 123.0 ppm). PBG prepared from a mixture of (4-/sup 13/C)ALA and (/sup 15/N)ALA was used to assign the 121.0 and 123.0 ppm resonances to C/sub 5/ and C/sub 3/, respectively. For the enzyme-bound equilibrium complex formed from holoenzyme and (4-/sup 13/C)ALA, two peaks of equal area with chemical shifts of 121.5 and 127.2 ppm are observed (line widths approx.more » 50 Hz), indicating that the predominant species is probably a distorted form of PBG. When excess free PBG is present, it is in slow exchange with bound PBG, indicating an exchange rate of < 10 s/sup -1/, which is consistent with the turnover rate of the enzyme. For the complex formed from (4-/sup 13/C)ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formation, the predominant species is a Schiff base adduct (chemical shift = 166.5 ppm, line width approx. 50 Hz). Free ALA is in slow exchange with the Schiff base. Activation of the MMTS-modified enzyme-Schiff base complex with /sup 113/Cd and 2-mercaptoethanol results in the loss of the Schiff base signal and the appearance of bound PBG with the same chemical shifts as for the bound equilibrium complex with Zn(II) enzyme. Neither splitting nor broadening from /sup 113/Cd-/sup 13/C coupling was observed.« less
  • γ-Glutamyl transpeptidase 1 (GGT1) is a cell surface, N-terminal nucleophile hydrolase that cleaves glutathione and other γ-glutamyl compounds. GGT1 expression is essential in cysteine homeostasis, and its induction has been implicated in the pathology of asthma, reperfusion injury, and cancer. In this study, we report four new crystal structures of human GGT1 (hGGT1) that show conformational changes within the active site as the enzyme progresses from the free enzyme to inhibitor-bound tetrahedral transition states and finally to the glutamate-bound structure prior to the release of this final product of the reaction. The structure of the apoenzyme shows flexibility within themore » active site. The serine-borate-bound hGGT1 crystal structure demonstrates that serine-borate occupies the active site of the enzyme, resulting in an enzyme-inhibitor complex that replicates the enzyme's tetrahedral intermediate/transition state. The structure of GGsTop-bound hGGT1 reveals its interactions with the enzyme and why neutral phosphonate diesters are more potent inhibitors than monoanionic phosphonates. These structures are the first structures for any eukaryotic GGT that include a molecule in the active site covalently bound to the catalytic Thr-381. The glutamate-bound structure shows the conformation of the enzyme prior to release of the final product and reveals novel information regarding the displacement of the main chain atoms that form the oxyanion hole and movement of the lid loop region when the active site is occupied. Lastly,tThese data provide new insights into the mechanism of hGGT1-catalyzed reactions and will be invaluable in the development of new classes of hGGT1 inhibitors for therapeutic use.« less
  • /sup 13/C NMR has been used to observe the equilibrium complex of (5,5-/sup 2/H,5-/sup 13/C)-5-aminolevulinate ((5,5-/sup 2/H,5-/sup 13/C)ALA) bound to porphobilinogen (PBG) synthase (5-aminolevulinate dehydratase), a 280,000-dalton protein. (5,5-/sup 2/H,5-/sup 13/C)ALA (chemical shift 46.9 ppm in D/sub 2/O) was prepared from (5-/sup 13/C)ALA through enolization in deuteriated neutral potassium phosphate buffer. In the PBG synthase reaction (5,5-/sup 2/H,5-/sup 13/C)ALA forms (2,11,11-/sup 2/H,2,11-/sup 13/C)PBG (chemical shifts 116.2 ppm for C/sub 2/ and 34.2 ppm for C/sub 11/ in D/sub 2/O). For the complex formed between (5,5-/sup 2/H,5-/sup 13/C)ALA and methyl methanethiosulfonate (MMTS) modified PBG synthase, which does not catalyze PBG formationmore » but can form a Schiff base adduct, the chemical shift of 44.2 ppm (line width 92 Hz) identifies and imine structure as the predominant tautomeric form of the Schiff base. By comparison to model compounds, the stereochemistry of the imine has been deduced; however, the protonation state of the imine nitrogen remains unresolved. Reconstitution of the MMTS-modified enzyme-Schiff base complex with Zn(II) and 2-mercaptoethanol results in the holoenzyme-bound equilibrium complex; this complex contains predominantly enzyme-bound PBG, and spectra reveal two peaks from bound PBG and two from free PBG. For bound PBG, C/sub 2/ is -2.8 ppm from the free signal and C/sub 11/ is +2.6 ppm from the free signal; the line widths of the bound signals are 55 and 75 Hz, respectively. To aid in interpretation of these shifts, the /sup 13/C NMR chemical shifts of PBG were investigated as functions of pH and a variety of organic solvents. The observed shifts of bound PBG are not consistent with simple protonation/deprotonation of PBG nor with changes that can be duplicated by solvation by simple organic solvents.« less