Differential inhibition of tryptophan synthase and of tryptophanase by the two diastereoisomers of 2,3-dihydro-L-tryptophan. Implications for the stereochemistry of the reaction intermediates

Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan, which are analogs of a proposed reaction intermediate, are potent competitive inhibitors of both tryptophanase and the alpha 2 beta 2 complex of tryptophan synthase (Phillips, R. S., Miles, E. W., and Cohen, L. A. (1984) Biochemistry 23, 6228-6234). Since these inhibitors can exist in two diastereoisomeric forms, which we expected to differ in inhibitory potency, we have separated the diastereoisomers of 2,3-dihydro-L-tryptophan by preparative high performance liquid chromatography. These diastereoisomers were designated "A" and "B" in order of elution from the high performance liquid chromatography column. Diastereoisomer B is a potent competitive inhibitor of the alpha 2 beta 2 complex of tryptophan synthase with KI = 6 microM at pH 7.8 and 25 degrees C. In contrast, diastereoisomer A is a weak competitive inhibitor, with KI = 940 microM under these conditions. With tryptophanase, the situation is reversed; diastereoisomer A is a potent slow-binding competitive inhibitor of tryptophanase with KI = 2 microM at pH 8.0 and 25 degrees C, while diastereoisomer B is much weaker with KI = 1600 microM under these conditions. These results not only provide additional support for the proposal that the indolenine tautomer of tryptophan is an intermediate in the reactions catalyzed by both enzymes but also suggest that these enzymes catalyze their respective reactions via enantiomeric indolenine intermediates.     

L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.     

Isolation of salt-soluble elastin from ligamentum nuchae of copper-deficient calf

This paper describes the isolation and amino acid analysis of un-cross-linked elastin obtained by neutral salt extraction from the ligamentum nuchae of a calf fed from birth to 9 months on a diet low in copper.     

Hg ++ - a DCMU independent electron acceptor of photosystem II

Mercuric chloride functions as a direct electron acceptor from the quencher of fluorescence in Photosystem II. The photoreduction of ferricyanide, dichlorophenol-indophenol or methyl viologen is inhibited by mercuric ion while oxygen evolution is uneffected. Mercuric chloride supported oxygen evolution (mercury Hill reaction) is not prevented by DCMU or other similar electron transport inhibitors.     

The use of peptide analogs with improved stability and MHC binding capacity to inhibit antigen presentation in vitro and in vivo

The identification of a core region for OVA 323-339, which is critical in determining binding to IAd, has enabled us to generate a series of analog peptides in which this core region was extended at both the N and C termini with different amino acid residues. When assessed for binding capacity, several peptides were shown to have increased affinity for IAd compared with the parent sequence, and in addition, some peptides had acquired binding specificities for class II MHC haplotypes not present for OVA 323-339. These peptides were next examined for their ability to inhibit T cell responses in vitro and in vivo. The correlation between binding and the ability to inhibit T cell activation in vitro was good. However, when assessed in vivo, it was clear that high Ia binding was not sufficient in itself to define the inhibitory capacity of a given peptide. That this discrepancy was due to differences in degradation of the core-extended peptides was suggested by 1) results from an inhibition of Ag presentation assay, in which the pulse period with Ag and inhibitor was extended to 20 h; and 2) direct analysis of peptide stability by using reverse phase HPLC. Finally, by protecting the peptide from degradation with N- and C-terminal substitutions of D-amino acids, the inhibitory capacity of an unstable core-extended peptide in vitro could be greatly enhanced. These data indicate that the core extension approach may be one method by which antagonists for MHC class II molecules may be generated.