(1) Conformational heterogeneity about proline and pipecolic acid peptide bonds. (2) Structure of substrate analogs bound to enoyl-CoA hydratas

The factors which affect the cis-trans isomerization about proline and about pipecolic peptide bonds were studied by NMR spectroscopy using model peptides. In the second system, the structure of substrate analogs, both free and bound to the enzyme enoyl-CoA hydratase, were determined using homonuclear and heteronuclear multidimensional NMR spectroscopy.;In an analysis of the protein structures in the protein data bank by Steward and coworkers, they found that there is a marked tendency for an aromatic amino acid to precede a cis proline residue. There are also significant differences between the three aromatic amino acids with Tyr exhibiting a noticeably higher propensity than Phe or Trp to precede a cis proline residue. It is not known if this preference is due to a specific local interaction between an aromatic residue and a proline. In order to study the role which local interactions play in these conformation preferences, a set of tetrapeptides of the general sequence Acetyl-Gly-X-Pro-Gly-Carboxamide (GXPG) where X = Tyr, Phe, Trp, Ala or cyclohexyl alanine (Cha), were synthesized and studied by NMR spectroscopy. Analysis of the NMR data indicates that cis prolyl peptide bonds are stabilized by aromatic-proline interactions.;Pipecolic acid (Pip) is a proline analog which contains a six-membered hexahydropyridine ring. It is found in several important natural products including immunosuppresants and has been extensively used as a proline substitute in numerous syntheses of peptidomimetics. Surprisingly little is known about the thermodynamics and the kinetics of cis trans isomerization about pipecolic peptide bonds. A set of peptides of the sequence AcetylGly-X-Pip-Gly-Carboxamide (X = A, F, Y, W or Cha) were prepared. The conformational properties of the peptides and the thermodynamics and kinetics of the isomerization about the pipecolic peptide bonds were analyzed using NMR methods. The substitution of a pipecolic residue for a proline leads to (1) a significant increase of the population of the cis conformer, (2) a reduction in the Van't Hoff enthalpy for isomerization and (3) acceleration of the rates of isomerization.;The determination of the structure of the substrate analog 2,4-hexadienoyl-coenzyme A (HD-CoA) bound to the enzyme enoyl-CoA hydratase using transferred nuclear Overhauser enhancement (TRNOE) spectroscopy is described. The conformation of the CoA portion of bound HD-CoA is strikingly similar to that of the CoA portion of 4-hydroxybenzoyl-CoA bound to the active site of 4-chlorobenzoyl-CoA dehalogenase. This finding leads to the use of homology modeling to develop a model of the active site of the enzyme.;The stereospecific1H and 13C resonance assignments and the conformational analysis of hexadienoyl-CoA (HD-CoA) and crotonyl-CoA (Cr-CoA) are described. The two diastereotopic methyl groups at the C2'' carbon were assigned via transferred nuclear Overhauser effect experiments on the complex of HD-CoA with the enzyme enoyl-CoA hydratase. The two diastereotopic 1' ' protons were assigned using heteronuclear multiple-bond correlation experiments in conjunction with rotating frame nuclear Overhauser spectroscopy. These stereospecific assignments allow the rotamer populations about the C1''-C2' ' bond and the C2' '-C3'' bonds to be analyzed for the first time.