Beta-Endoglucanase from Pyrococcus furiosus.
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Amino Acid Sequence↗
Biomedical subjects
S G Cady
Publications and source records attributed to S G Cady.
1-Methyl-DL-tryptophan, beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of tryptophan), and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of tryptophan) are competitive inhibitors for indoleamine 2,3-dioxygenase.
1-methyl-DL-Trp, beta-(3-benzofuranyl)-DL-alanine (the oxygen analog of Trp), and beta-[3-benzo(b)thienyl]-DL-alanine (the sulfur analog of Trp), each of which has a substitution at the indole nitrogen atom, were found to be the first examples of potent substrate analog competitive inhibitors (Ki 7-70 microM) with respect to the substrates D-Trp and L-Trp for rabbit small intestinal indoleamine 2,3-dioxygenase. Binding studies using optical absorption and CD spectroscopy demonstrated that these three inhibitors cause spectral changes upon binding to the native ferric, ferrous, ferrous-CO, and ferrous-NO enzymes. Such spectral effects of 1-methyl-DL-Trp on all of these enzyme derivatives were similar to those caused by L-Trp, while the sulfur and the oxygen analogs of Trp exhibit relatively small effects except for those observed for the sulfur analog with CD spectroscopy. Each of these three Trp analog inhibitors competes with L-Trp for the ferrous-CO enzyme, a model for the ferrous-O2 enzyme. The present findings demonstrate that, although substitution of a methyl group for the hydrogen atom on the indole nitrogen or of a more electron-inductive sulfur or oxygen atom for the indole nitrogen atom does not prevent the binding of the resulting Trp analog to indoleamine 2,3-dioxygenase, the free form of the indole nitrogen base is an important physical and/or electronic structural requirement for Trp to be metabolized by the enzyme. The inability of 1-methyl-Trp to serve as a substrate for the dioxygenase supports a view that singlet oxygen is not the reactive oxygen species involved in the dioxygenation of Trp by the enzyme.
Alanine↗
Enzyme kinetic and spectroscopic studies of inhibitor and effector interactions with indoleamine 2,3-dioxygenase. 1. Norharman and 4-phenylimidazole binding to the enzyme as inhibitors and heme ligands.
The effects of norharman, one of the few known inhibitors of the heme protein indoleamine 2,3-dioxygenase, and of 4-phenylimidazole (4-PheImid), a heme ligand, on the catalytic (Vmax, Km) and spectroscopic properties (optical absorption, CD, and magnetic CD) of the rabbit small intestinal dioxygenase were investigated. Assays were performed with the substrate L- or D-tryptophan (Trp) and an ascorbic acid-methylene blue cofactor system at 25 degrees C. This study has revealed that both norharman and 4-PheImid exhibit noncompetitive inhibition with respect to L-Trp and D-Trp. The binding of norharman to the enzyme results in the formation of a low-spin complex in both the ferric and ferrous enzyme with comparable dissociation constants (Kd = approximately 10 microM at pH 7.0) that are about 10 times smaller than the observed Ki value. L-Trp exerts no effect for the ferric enzyme and slight negative cooperative effects for the ferrous enzyme on norharman binding. Close spectral similarities are observed between the adducts of the enzyme with norharman and 4-PheImid in the respective oxidation states. This, together with competition experiments using cyanide, demonstrates that norharman binds directly to the heme iron of the enzyme as a nitrogen donor ligand. Thus, norharman competes with O2 for the heme iron of the ferrous (active) enzyme, resulting in the observed inhibition. L-Trp and 4-PheImid appear to compete for the heme binding site in the ferric enzyme and display slight negative cooperativity on binding to the ferrous enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Alkaloids↗
Identification of fibroblasts as a major site of albumin catabolism in peripheral tissues.
Rat serum albumin has been labeled with dilactitol-125I-tyramine, (125I-DLT) a radioactive tracer which remains entrapped within lysosomes following cellular uptake and degradation of the carrier protein. Similar kinetics of clearance from the rat circulation were observed for albumin labeled conventionally with 125I or 125I-DLT-albumin, both proteins having circulating half-lives of approximately 2.2 days. In contrast, the recovery of whole body radioactivity had half-lives of approximately 2.2 and 5.1 days, respectively, for the two protein preparations, indicating substantial retention of degradation products derived from catabolism of 125I-DLT-albumin. Measurement of total and acid-soluble radioactivity in tissues 2 or 4 days after injection of 125I-DLT-albumin revealed that skin and muscle accounted for the largest fraction (50-60%) of degradation products in the body. Fibroblasts were identified by autoradiography as the major cell type containing radioactive degradation products in skin and muscle. Fibroblasts were isolated from skin by collagenase digestion, followed by density gradient centrifugation. The amount of acid-soluble radioactivity recovered in these cells was in excellent agreement with that predicted based on acid precipitation of solubilized whole skin preparations. These studies demonstrate for the first time that fibroblasts are a major cell type involved in the degradation of albumin in vivo.
Animals↗