Recombinant hirudin: kinetic mechanism for the inhibition of human thrombin

Recombinant hirudin variant-2(Lys47 ), was found to be a competitive inhibitor of human α-thrombin with respect to peptidyl p-Miitroanilide substrates. These results contrast with those of Degryse and coworkers that suggest that recombinant hirudin variant-2(Lys47) inhibited thrombin by a non-competitive mechanism [Degryse et al. (1989) Protein Engng, 2, 459-465], γ-Thrombin, which can arise from α-thrombin by autolysis, was shown to have an affinity for recombinant hirudin variant-2(Lys47) that was four orders of magnitude lower than that of α-thrombin. It was demonstrated that the apparent noncompetitive mechanism observed previously was probably caused by a contamination of the thrombin preparation by γ-thrombin. Comparison of the inhibition of α-thrombin by recombinant hirudins variant-2(Lys47) and variant-1, which differ from one another in eight out of 65 amino acids, indicated that the two variants have essentially the same kinetic parameter

Recombinant human interleukin-12 is the second example of a C-mannosylated protein

The β-chain of human interleukin 12 (IL-12) contains at position 319-322, the sequence Trp-x-x-Trp. In human RNase 2 this is the recognition motif for a new, recently discovered posttranslational modification, i.e., the C-glycosidic attachment of a mannosyl residue to the side chain of tryptophan. Analysis of C-terminal peptides of recombinant IL-12 (rHuIL-12) by mass spectrometry and NMR spectroscopy revealed that Trp-319β is (partially) C-mannosylated. This finding was extended by in vitro mannosylation experiments, using a synthetic peptide derived from the same region of the protein as an acceptor. Furthermore, human B-lymphoblastoid cells, which secrete IL-12, were found to contain an enzyme that carries out the C-mannosylation reaction. This shows that nonrecombinant IL-12 is potentially C-mannosylated as well. This is only the second report on a C-mannosylated protein. However, the occurrence of the C-mannosyltransferase activity in a variety of cells and tissues, and the presence of the recognition motif in many proteins indicate that more C-mannosylated proteins may be foun

P-hydroxybenzoate hydroxylase. Determination of the amino acid sequence and its integration with the crystal structure.

Het enzym p-hydroxybenzoaat hydroxylase katalyseert een belangrijke reaktie in de metabolische weg voor de afbraak van aromatische verbindingen (B-ketoadipaat route). […] Het enzym bevat flavine adenine dinucleotide (FAD). De flavine wordt tijdens de reaktie gereduceerd door NADPH, waarna het een covalent complex vormt met zuurstof. Eén van de zuurstofatomen wordt overgedragen aan het substraat, terwijl het andere wordt afgesplitst in de vorm van water. ... Zie: Samenvatting

Protein C-mannosylation: Facts and questions.

Among the posttranslational modifications of proteins, glycosylation is probably the most abundant one. Two main types of protein glycosylation have been known for several years, namely N-glycosylation and O-glycosylation. Their biochemical properties, structure and biosynthesis, have been described extensively. Their biological functions are also known for a number of proteins, although in many cases the function remains speculative despite continuous efforts. A few years ago, a new type of protein glycosylation was found, which is different from the above-mentioned ones. It was called C-glycosylation, since the sugar is linked to the protein through a carbon-carbon bond. This article reviews the biochemistry of C-glycosylation, the biosynthetic pathway and structural requirements. Possible biological functions of this modification are also discussed

P - hydroxybenzoate hydroxybase Determination of the anmino acid sequence and its integrationwith the crystal structure

SIGLEBSE B210283X / UCL - Université Catholique de LouvainBEBelgiu