Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.3.3.1 (citrate synthase)
 4,488 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ionization state and hydrogen bonding environment of the transition state analogue (TSA) inhibitor, carboxymethyldethia coenzyme A (CMX), bound to citrate synthase have been investigated using solid state NMR. This enzyme-inhibitor complex has been studied in connection with the postulated contribution of short hydrogen bonds to binding energies and enzyme catalysis: the X-ray crystal structure of this complex revealed an unusually short hydrogen bond between the carboxylate group of the inhibitor and an aspartic acid side chain [Usher et al. (1994) Biochemistry 33, 7753-7759]. To further investigate the nature of this short hydrogen bond, low spinning speed 13C NMR spectra of the CMX-citrate synthase complex were obtained under a variety of sample conditions. Tensor values describing the chemical shift anisotropy of the carboxyl groups of the inhibitor were obtained by simulating MAS spectra (233 +/- 4, 206 +/- 5, and 105 +/- 2 ppm vs TMS). Comparison of these values with our previously reported database and ab initio calculations of carbon shift tensor values clearly indicates that the carboxyl is deprotonated. New data from model compounds suggest that hydrogen bonds in a syn arrangement with respect to the carboxylate group have a pronounced effect upon the shift tensors for the carboxylate, while anti hydrogen bonds, regardless of their length, apparently do not perturb the shift tensors of the carboxyl group. Thus the tensor values for the enzyme-inhibitor complex could be consistent with either a very long syn hydrogen bond or an anti hydrogen bond; the latter would agree very well with previous crystallographic results. Two-dimensional 1H-13C heteronuclear correlation spectra of the enzyme-inhibitor complex were obtained. Strong cross-peaks were observed from the carboxyl carbon to proton(s) with chemical shift(s) of 22 +/- 5 ppm. Both the proton chemical shift and the intensity of the cross-peak indicate a very short hydrogen bond to the carboxyl group of the inhibitor, the C.H distance based upon the cross-peak intensity being 2.0 +/- 0.4 A. This proton resonance is assigned to Hdelta2 of Asp 375, on the basis of comparison with crystal structures and the fact that this cross-peak was absent in the heteronuclear correlation spectrum of the inhibitor-D375G mutant enzyme complex. In summary, our NMR studies support the suggestion that a very short hydrogen bond is formed between the TSA and the Asp carboxylate.
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PMID:Solid state NMR studies of hydrogen bonding in a citrate synthase inhibitor complex. 1038 46

Acetobacter aceti converts ethanol to acetic acid, and strains highly resistant to both are used to make vinegar. A. aceti survives acetic acid exposure by tolerating cytoplasmic acidification, which implies an unusual adaptation of cytoplasmic components to acidic conditions. A. aceti citrate synthase (AaCS), a hexameric type II citrate synthase, is required for acetic acid resistance and, therefore, would be expected to function at low pH. Recombinant AaCS has intrinsic acid stability that may be a consequence of strong selective pressure to function at low pH, and unexpectedly high thermal stability for a protein that has evolved to function at approximately 30 degrees C. The crystal structure of AaCS, complexed with oxaloacetate (OAA) and the inhibitor carboxymethyldethia-coenzyme A (CMX), was determined to 1.85 A resolution using protein purified by a tandem affinity purification procedure. This is the first crystal structure of a "closed" type II CS, and its active site residues interact with OAA and CMX in the same manner observed in the corresponding type I chicken CS.OAA.CMX complex. While AaCS is not regulated by NADH, it retains many of the residues used by Escherichia coli CS (EcCS) for NADH binding. The surface of AaCS is abundantly decorated with basic side chains and has many fewer uncompensated acidic charges than EcCS; this constellation of charged residues is stable in varied pH environments and may be advantageous in the A. aceti cytoplasm.
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PMID:Structure of a NADH-insensitive hexameric citrate synthase that resists acid inactivation. 1708 2