Isoleucine, protonated

Fig. 5. 500 MHz HRMAS H NMR spectra of FMOC-isoleucine on Wang resin swollen in DMF- 7 and spun at 4 kHz. Spectrum A shows the presence of dissolved phenylalanine peaks, particularly around 3.0 ppm from its methylene protons, and residual protons of the solvent. Application of a diffusion filter using gradients selectively removes these signals in spectrum B. Reproduced with permission from Ref. 50. Copyright 1999 Elsevier.

The resonances between —0.2 and 0.7 ppm could be shifted to their high field positions if the corresponding protons were located near the heme outside the area of the axial ligands (Fig. 7), or near any one of the aromatic amino acids. McDonald et al. (79) tentatively assigned the three methyl resonances to the threonyl residue at position 19 of the polypeptide chain, and to isoleucine-81. 

Pyrrolizidine Alkaloids.—The necic acid component of senecionine (8) derives from two molecules of isoleucine, radioactivity from precursor amino-acid being equally incorporated into both halves of the necic acid fragment, as shown in Scheme 2 (c/. Vol. 9, p. 4). It has now been shown that biotransformation of isoleucine into the necic acid involves loss of half of a tritium label from C-4 in each of the two amino-acid fragments.6 Removal of a proton is, therefore, stereospecific, and oxidation at C-4 does not proceed beyond the two-electron level i.e., a higher intermediate oxidation level, corresponding to a ketone, is excluded. Further results indicate that for each molecule of isoleucine it is the 4-pro-S proton [see (14)] which is lost. 

Exhaustive NMR spectral studies have been reported for ceanothine-B(i9, 24-26), for pandamine (17), for americine (20), for aralionine-A (31), and their derivatives. The number and type of NMe and OMe groups can be learned as can the number and position of the amide NH protons even though exchange with deuterium is not always straightforward. In examples in which mass spectra do not differentiate between leucine and isoleucine the NMR spectra sometimes can do so. 

An apparently similar, antiperiplanar elimination with loss of the isoleucine 4-pro-S proton, to generate a double-bond of opposite geometry in alkaloidal necic acid fragments, has been observed13 (cf. Vol. 11, p. 2). 

A similar stereochemical question as in the /8-replacement reactions can be asked in the a, /8-eliminations where the group X is replaced by a hydrogen, i.e., is the proton added at C-/8 of the PLP-aminoacrylate on the same face from which X departed or on the opposite face This question has been answered for a number of enzymes which generate either a-ketobutyrate or pyruvate as the keto acid product. Crout and coworkers [119,120] determined the steric course of proton addition in the a,/8-elimination of L-threonine by biosynthetic L-threonine dehydratase and of D-threonine by an inducible D-threonine dehydratase, both in Serratia marcescens. Either substrate, deuterated at C-3, was converted in vivo into isoleucine, which was compared by proton NMR to a sample prepared from (3S)-2-amino[3-2H]butyric acid. With both enzymes the hydroxyl group at C-3 was replaced by a proton in a retention mode. Although this has not been established with certainty, it is likely that both enzymes, like other bacterial threonine dehydratases [121], contain PLP as cofactor. Sheep liver L-threonine dehydratase, on the other hand, is not a PLP enzyme but contains an a-ketobutyrate moiety at the active site [122], It replaces the hydroxyl group of L-threonine with H in a retention mode, but that of L-allothreonine in an inversion mode [123]. Snell and coworkers [124] established that the replacement of OH by H in the a, /8-elimination of D-threonine catalyzed by the PLP-containing D-serine dehydratase from E. coli also proceeds in a retention mode. They... 

Masuko, T., Kashiwagi, K., Kuno, T., Nguyen, N. D., Pahk, A. J., Fukuchi, J., Igarashi, K., and Williams, K. (1999). A regulatory domain (R1-R2) in the amino terminus of the N-methyl-D-aspartate receptor Effects of spermine, protons, and ffenprodil, and structural similarity to bacterial leucine/isoleucine/valine binding protein. Mol. Pharmacol. 55, 957-969. 

This enzyme s role in humans is to assist the detoxification of propionate derived from the degradation of the amino acids methionine, threonine, valine, and isoleucine. Propionyl-CoA is carboxylated to (5 )-methylmalonyl-CoA, which is epimerized to the (i )-isomer. Coenzyme Bi2-dependent methylmalonyl-CoA mutase isomerizes the latter to succinyl-CoA (Fig. 2), which enters the Krebs cycle. Methylmalonyl-CoA mutase was the first coenzyme B -dependent enzyme to be characterized crystallographically (by Philip Evans and Peter Leadlay). A mechanism for the catalytic reaction based on ab initio molecular orbital calculations invoked a partial protonation of the oxygen atom of the substrate thioester carbonyl group that facilitated formation of an oxycyclopropyl intermediate, which connects the substrate-derived and product-related radicals (14). The partial protonation was supposed to be provided by the hydrogen bonding of this carbonyl to His 244, which was inferred from the crystal structure of the protein. The ability of the substrate and product radicals to interconvert even in the absence of the enzyme was demonstrated by model studies (15). 

Figure 18 SOS-NMR using deuterated and selectively protonated protein can resolve ambiguity of ligand orientation, (a) Bottom 1 D H NMR reference spectrum that shows the resonances of the ligand SB203580. Middle 1 D H STD spectrum recorded in the presence of p38a. Top 1D H STD spectrum recorded of a sample prepared according to the SOS procedure of perdeuterated kinase and unlabeled isoleucine residues. A pronounced STD effect for the HI and H4 protons and only weak effects for the H6 and H5 protons resolves ambiguity of ligand orientation in the binding pocket, (b) Distance analysis of the reference X-ray structure.

The newly formed amino-terminal group of isoleucine 16 turns inward and forms an ionic bond with aspartate 194 in the interior of the chymotrypsin molecule (Figure 10.33). Protonation of this amino group stabilizes the active form of chymotrypsin. 

Proton resonance spectra of denatured proteins consist of sharp peaks which correspond to a summation of resonances from individual residues [129]. In C spectra of denatured proteins, it is possible to distinguish all the carbon resonances of the aromatic side chains of histidine, phenylalanine, tyrosine and tryptophan, and separate resonances from alanine, arginine, glycine, isoleucine, leucine, threonine, valine and occasionally methionine [130]. (A natural abundance C spectrum of a 13 mM solution of lysozyme takes only 4 hr accumulation time using 20 mm sample tubes [131]). 

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