Identification of the C2‐1H histidine NMR resonances in chloramphenicol acetyltransferase by a 13C‐1H heteronuclear multiple quantum coherence method

Derrick, Jeremy P.; Lian, Lu‐Yun; Roberts, G. C. K.; Shaw, William V.

doi:10.1016/0014-5793(91)80219-s

Cited by 6 publications

(3 citation statements)

References 14 publications

(1 reference statement)

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“…Using n.m.r. techiques, Derrick et al (1991) attempted to determine directly the microscopic pKa of His-195 by titrating the histidine residues of CATIII. However, a signal for His-195 was not among the six C-2 proton resonances that were observed over the pH range 6-8.…”

Section: Discussionmentioning

confidence: 99%

The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

Lewendon

Shaw

1993

Biochemical Journal

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A catalytically essential histidine residue (His-195) of chloramphenicol acetyltransferase (CAT) acts as a general base in catalysis, abstracting a proton from the primary hydroxy group of chloramphenicol. The pKa of His-195 has been determined from the pH-dependence of chemical modification. Both methyl 4-nitrobenzenesulphonate and iodoacetamide inactivate CAT by irreversible modification of His-195. The kinetics of inactivation by methyl 4-nitrobenzenesulphonate are pseudo-first-order, and the pH-dependence of inactivation yields a pKa value of 6.60. Iodoacetamide inactivation proceeds with second-order kinetics and a pKa value of 6.80. An alternative site of modification at the active site of CAT is the thiol group of Cys-31, a residue which has no catalytic role. On replacement of Cys-31 with alanine (Ala-31 CAT), the pH-dependence of iodoacetamide inactivation gives a pKa value of 6.66. The pKa values derived from chemical-modification experiments directed at His-195 are in agreement with the pKa values of 6.62 and 6.61 determined for wild-type and Ala-31 CAT respectively from the pH-dependence of kcat/Km.

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Section: Discussionmentioning

confidence: 99%

The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

Lewendon

Shaw

1993

Biochemical Journal

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“…NMR Methods. All samples of CATm for NMR were transferred into 50 mM sodium phosphate buffer in D20 (pH* 7.5) as described by Derrick et al (1991). For the 1-D and 2-D 13C (<v2) half-filtered NOESY experiments, the concentration of 1,3-[2-13C] -diacetylchloramphenicol was equal to that of CAT subunits, approximately 2 mM l,3-[2-13C]diacetylchloramphenicol and 50 mg/mL protein.…”

Section: Methodsmentioning

confidence: 99%

“…1992 American Chemical Society The NMR spectra of this enzyme are too complex to analyze by conventional NMR methods. An alternative approach is to use stable isotope labeling, such as selective deuteration (Markley et al, 1968;LeMaster, 1989) or I3C or l5N labeling in conjunction with "isotope-editing" methods (Derrick et al, 1991; Otting & Wflthrich, 1990; Griffey & Redfield, 1987), to simplify the spectra. 13C-or 15Nlabeled ligands can be employed to study the conformation of the bound species and its contacts with neighboring amino acid residues [e.g., Fesik et al (1988)].…”

Section: Non-enzymic Rearrangement O Acmentioning

confidence: 99%

Analysis of the binding of 1,3-diacetylchloramphenicol to chloramphenicol acetyltransferase by isotope-edited proton NMR and site-directed mutagenesis

Derrick¹,

Lian²,

Roberts³

et al. 1992

Biochemistry

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The binary complex of diacetylchloramphenicol and chloramphenicol acetyltransferase (CAT) has been studied by a combination of isotope-edited 1H NMR spectroscopy and site-directed mutagenesis. One-dimensional HMQC spectra of the complex between 1,3-[2-13C]diacetylchloramphenicol and the type III natural variant of CAT revealed the two methyl 1H signals arising from each 13C-labeled carbon atom in the acetyl groups of the bound ligand. Slow hydrolysis of the 3-acetyl group by the enzyme precluded further analysis of this binary complex. It was possible to slow down the rate of hydrolysis by use of the catalytically defective S148A mutant of CATIII (Lewendon et al., 1990); in the complex of diacetylchloramphenicol with S148A CATIII, the chemical shifts of the acetyl groups of the bound ligand were the same as in the wild-type complex. The acetyl signals were individually assigned by repeating the experiment using 1-[2-13C],3-[2-12C]diacetylchloramphenicol, where only one signal from the bound ligand was observed. A two-dimensional 1H, 1H NOESY experiment, with 13C(omega 2) half-filter, on the 1,3-[2-13C]diacetylchloramphenicol/S148A CATIII complex showed a number of intermolecular NOEs from each methyl group in the ligand to residues in the chloramphenicol binding site. The 3-acetyl group showed strong NOEs to two aromatic signals which were selected for assignment. The possibility that the NOEs originated from the aromatic protons of diacetylchloramphenicol itself was eliminated by assignment of the signals from enzyme-bound diacetylchloramphenicol and chloramphenicol using perdeuterated CATIII. Examination of the X-ray crystal structure of the chloramphenicol/CATIII binary complex indicated four plausible candidate aromatic residues: Y25, F33, F103, and F158.(ABSTRACT TRUNCATED AT 250 WORDS)

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NMR Studies of Enzyme-Substrate and Protein-Protein Interactions

Roberts

1996

Dynamics and the Problem of Recognition in Biological Macromolecules

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Identification of the C2‐¹H histidine NMR resonances in chloramphenicol acetyltransferase by a ¹³C‐¹H heteronuclear multiple quantum coherence method

Cited by 6 publications

References 14 publications

The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase

Analysis of the binding of 1,3-diacetylchloramphenicol to chloramphenicol acetyltransferase by isotope-edited proton NMR and site-directed mutagenesis

NMR Studies of Enzyme-Substrate and Protein-Protein Interactions

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