Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide.

Metal insertion into an engineered cytoplasmic form of the multicopper enzyme N2O reductase (N2OR) (EC 1.7.99.6) of Pseudomonas stutzeri was studied. The reductase has an unusually long presequence of 50 amino acids for translocation into the periplasm. The signal peptide of N2OR shares a conserved twin-arginine sequence motif with the signal peptides of other N2O reductases and a sizeable group of periplasmic or membrane-bound enzymes, requiring cofactor insertion or processing. A catalytically inactive reductase, N2ORR20D, that lacked Cu, accumulated in the cytoplasm on mutation of the first arginine of this motif. The CuA site of N2ORR20D could be reconstituted in vitro indicating that the lack of metal was not due to a serious conformational restraint. Our findings locate the event of in vivo Cu insertion into N2OR in the periplasm or allow it to take place concomitant with protein translocation.

Amino acid sequence determination of human S100A12 (P6, calgranulin C, CGRP, CAAF1) by tandem mass spectrometry.

S100A12 has been isolated from human neutrophils. The molecular weight and the amino acid sequence of S100A12 was determined by electrospray-mass spectrometry, tandem mass spectrometry and Edman microsequence analysis. Interestingly, a sequence comparison of S100A12 with all known human S100 proteins revealed that S100A12 is the most divergent of the S100 proteins.

Amino acid sequence analysis of human S100A7 (psoriasin) by tandem mass spectrometry.

The Ca(2+)-binding proteins regulate a number of cellular and extracellular activities and deregulations of S100 gene expression are associated with several human diseases. For example, S100A7 is upregulated in psoriatic skin, implicating a link with psoriasis, a chronic inflammatory dermatosis. We purified human S100A7 and determined its protein sequence by tandem mass spectrometry and Edman microsequence analysis. Interestingly, a sequence comparison of S100A7 with all known human S100 proteins showed that S100A7 is the most divergent of all S100 proteins.

6-Pyruvoyl tetrahydropterin synthase, an enzyme with a novel type of active site involving both zinc binding and an intersubunit catalytic triad motif; site-directed mutagenesis of the proposed active center, characterization of the metal binding site and modelling of substrate binding.

6-Pyruvoyl tetrahydropterin synthase (PTPS) is an enzyme involved in tetrahydrobiopterin biosynthesis, the cofactor for several aromatic amino acid monooxygenases and the nitric oxide synthases. The crystal structure of PTPS was recently solved and showed a homohexameric enzyme composed of a dimer of trimers. A transition metal binding site formed by the three histidine residues 23, 48 and 50 was found in each subunit. We showed by metal analysis and reconstitution of apo-PTPS that Zn(II) was the bound transition metal and responsible for the enzymatic activity. Site-directed mutagenesis of each of these three histidine residues resulted in a complete loss of metal binding and enzymatic activity. The three residues, Cys42, His89 and Glu133, located close to the metal binding site, were previously postulated to be involved in the catalytic reaction. We altered these residues and found a complete loss of enzymatic activity for the mutant C42A. The two mutants, H89N and E133Q, showed 4.3% and 1.3% enzymatic activity, respectively, but had similar KM values for the substrate as compared to wild-type PTPS. Based on these results we propose a model of the substrate fitted into the active site and we described a novel intersubunit catalytic triad motif composed of the amino acid residues Cys42, His89 and Asp88. Different from most other catalytic triads that catalyse the hydrolysis of an amide or ester bond, the catalytic triad in the active site of PTPS seems to be involved in the deprotonation of the substrate's side-chain carbons. Our model also proposes Zn(II) as the coordination site for the two substrate side-chain hydroxy groups as well as the involvement of Glu133 as putative stereospecific proton server.

Expression and characterization of recombinant human and rat liver 6-pyruvoyl tetrahydropterin synthase. Modified cysteine residues inhibit the enzyme activity.

6-Pyruvoyl-tetrahydropterin synthase is the rate-limiting enzyme in the synthesis of human tetrahydrobiopterin, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. The human and rat liver cDNAs encoding the 16-kDa subunit of 6-pyruvoyl tetrahydropterin synthase were expressed as maltose-binding-6-pyruvoyl-tetrahydropterin-synthase fusion proteins. After cleavage from the fusion protein, the human and rat enzymes were purified to homogeneity. Apparent Km for the substrate dihydroneopterin triphosphate (8.5 microM for the human and 8.0 microM for the rat enzyme), pI (4.6 and 4.8) and heat stability of the recombinant enzymes were similar to the native enzymes. The specific activity of the enzymes was enhanced up to fourfold in the presence of dithiothreitol during purification. The modification of the only cysteine residue in rat 6-pyruvoyl tetrahydropterin synthase, which is conserved in the human enzyme, inhibited its activity up to 80%. Modification under non-reducing conditions of both cysteine residues of the human enzyme by N-ethylpyridine resulted in a 95% loss of enzyme activity. This demonstrates that the two cysteines are not linked by disulfide bridges but rather involved in catalysis. Cross-linking experiments and analysis by gel electrophoresis showed predominantly trimeric and hexameric forms of the recombinant enzymes from both species suggesting that the native form is a homohexamer of 98 kDa, for the human, and 95 kDa, for the rat enzyme, composed of two trimeric subunits.

Mutants of human insulin-like growth factor II: expression and characterization of analogs with a substitution of TYR27 and/or a deletion of residues 62-67.

Five structural analogs of human insulin-like growth factor II (IGF II), [Leu27]IGF II, [Glu27]IGF II, des(62-67)IGF II, des(62-67)[Leu27]IGF II and des(62-67)[Glu27]IGF II were constructed by site-directed mutagenesis and expressed as protein A fusion proteins in E. coli BL21 pLysS cells, cleaved with CNBr and purified by affinity chromatography and HPLC. These mutants were tested for their binding affinities to type 1 and type 2 IGF receptors, to IGF binding protein-3 (IGFBP-3) and for their stimulation of thymidine incorporation into DNA. [Leu27]IGF II exhibits an affinity to the type 2 IGF receptor close to that of wild-type IGF II, but has lost completely the affinity to the type 1 IGF receptor. The results further suggest that the D domain, which is close to Tyr27, forms part of the binding region for the type 1 IGF receptor.

Mutants of human insulin-like growth factor II with altered affinities for the type 1 and type 2 insulin-like growth factor receptor.

With the aim to produce insulin-like growth factors (IGF) with enhanced specificity for the type 1 or type 2 IGF receptors, three mutants of IGF II have been prepared and expressed in NIH-3T3 cells. IGF II mutated at Tyr27 to Leu and Glu showed a 25- and 54-fold decrease in affinity for the type 1 IGF receptor and a 3.4- and 9.2-fold decrease in affinity for the type 2 IGF receptor. IGF II mutated at Phe48 to Glu showed a 18-fold decrease in affinity for the type 2 IGF receptor and a 2.8-fold decrease in affinity for the type 1 IGF receptor. These affinities were measured in radioreceptor assays using type 1 or 2 IGF receptor overexpressing cells. Data obtained on receptor cross-linking and thymidine incorporation assays confirmed the results of the radioreceptor assays. It is concluded that mutations of Tyr27 preferentially decrease binding to the type 1 IGF receptor and of Phe48 to the type 2 IGF receptor, either by the loss of a residue involved in receptor binding or by preferentially destabilizing the region involved in receptor binding.