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PubMed1980

Serine sensitivity of Escherichia coli K 12: partial characterization of a serine resistnat mutant that is extremely sensitive to 2-ketobutyrate.

E. coli wild type bacteria display sensitivity towards serine. A selection medium is described which allows selection of serine resistant mutants. One such mutant is described which presents pleiotropic alterations: it exhibits a thermosensitive growth pattern, alteration in the metabolism of the pppGpp and ppGpp nucleotides, cAMP intracellular level alteration, extreme sensitivity to 2-ketobutyric acid and a defect in the phosphotransferases permeation system. A conjecture explaining these apparently unrelated defects supposes that serine metabolism interferes via phosphoenol pyruvate with a cytoplasmic control of membrane activity (the mutant would be defective in the coupling between membrane and the protein responsible for its cytoplasmic control) and that 2-ketobutyrate is an effector of this activity.

Butyrates↗
PubMed1981

The role of serine hydroxymethyltransferase in cell proliferation: DNA synthesis from serine following mitogenic stimulation of lymphocytes.

It is shown that the time-course of incorporation of radioactivity from [3-14C]serine into nucleic acids parallels DNA synthesis following mitogenic stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA). The activity of serine hydroxymethyltransferase was elevated about four-fold in PHA-stimulated lymphocytes compared to that in unstimulated control cells. It is suggested that lymphocytes, in common with other proliferating cell systems, may synthesize serine de novo for utilization in pathways of nucleotide biosynthesis following mitogenic stimulation.

Cell Division↗
PubMed2004

Specificity comparison of a serine endopeptidase (SH1) and a serine thiol endopeptidase (STH2) purified from human urine.

In this study, we compared the properties of a serine endopeptidase H1 (SH1) and a serine thiol endopeptidase (STH2) purified from human urine by DEAE-cellulose followed by a Bio Gel A0.5 m or Sepharose Mercurial chromatographs. These enzymes differ in their action upon different hormone peptides. We used fluorogenic substrates to further characterize the enzyme. The substrate specificity of urinary SH1 was studied using different internally quenched fluorescent peptides, and AbzFGQEDDnp was hydrolyzed by SH1. Other enzymes present in urine, such as serine endopeptidase H2, prolyl endopeptidase, neutral endopeptidase like and angiotensin-I converting enzyme, were not able to hydrolyze this substrate. SH1 is 100% inhibited by PMSF and resistant to EDTA, OPA, thiorphan, E64, pOHMB and phosphoramidon. Endopeptidase STH2 is completely inhibited by PMSF, E64 and pOHMB. Enzyme SH1 hydrolyzes the peptide bound F5-S6 at bradykinin (BK: RPPGFSPFR) molecule and R-Q at AbzBKQEDDnp. When studying enzyme STH2, the cleavage sites determined to the related substrates were F5-S6 using BK as substrate and F-R using AbzBKQEDDnp. The kilometers value obtained for AbzBKQEDDnp and AbzFGQEDDnp were 1.18 and 0.007 uM, respectively. Kininases from kidney and urine can hydrolyze peptide bounds from components of the kallikrein-kinin system, the angiotensin-renin system and the neuropeptides system, straight contributing in kidney homeostasis. SH1 was located at the distal tubule [Casarini et al., 1999a, Am. J. Physiol. 277, F66] and can have an important function in the control of kinin found in this portion, since is known that all components of the kallikrein-kinin system were found in this portion. The physiological role of SHT2 could be related to the inter-relation between the kallikrein-kinin system and neuropeptides in the control of the water electrolyte balance [Braz. J. Med. Biol. Res. 25 (3) (1992) 219].

Amino Acid Sequence↗
PubMed2005

Epitope mapping of Burkholderia pseudomallei serine metalloprotease: identification of serine protease epitope mimics.

Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.

Amino Acid Sequence↗
PubMed1999

The inhibitory effect of 2-deoxyglucose on insulin receptor autophosphorylation does not depend on known serine phosphorylation sites or other conserved serine residues of the receptor beta-subunit.

Hyperglycemia induces insulin resistance in diabetic patients. It is known that supraphysiological levels of D-glucose or 2-deoxyglucose inhibit the insulin receptor and it is speculated that this effect is mediated by serine phosphorylation of the insulin receptor beta-subunit and other proteins of the insulin signaling chain. To test this hypothesis we prepared point mutations of the human insulin receptor where serine was exchanged to alanine at 16 different positions, either at known phosphorylation sites or at positions which are conserved in different tyrosine kinase receptors. These receptor constructs were expressed in HEK 293 cells and the effect of 2-deoxyglucose (25 mM) on insulin (100 nM) induced receptor autophosphorylation was studied. 2-Deoxyglucose consistently inhibits insulin stimulated autophosphorylation of all constructs to the same degree as observed in wild-type human insulin receptor. The data suggest that none of the chosen serine positions are involved in 2-deoxyglucose induced receptor inhibition.

Binding Sites↗
PubMed1989

Mechanism of interaction of O-amino-D-serine with sheep liver serine hydroxymethyltransferase.

The mechanism of interaction of O-amino-D-serine (OADS) with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in the enzyme activity, absorption spectra, circular dichroism (CD) spectra, and stopped-flow spectrophotometry. OADS was a reversible noncompetitive inhibitor (Ki = 1.8 microM) when serine was the varied substrate. The first step in the interaction of OADS with the enzyme was the disruption of enzyme-Schiff base, characterized by the rapid disappearance of absorbance at 425 nm (6.5 X 10(3) M-1 s-1) and CD intensity at 430 nm. Concomitantly, there was a rapid increase in absorbance and CD intensity at 390 nm. The spectral properties of this intermediate enabled its identification as pyridoxal 5'-phosphate (PLP). These changes were followed by a slow unimolecular step (2 X 10(-3) s-1) leading to the formation of PLP-OADS oxime, which was confirmed by its absorbance and fluorescence spectra and retention time on high-performance liquid chromatography. The PLP-OADS oxime was displaced from the enzyme by the addition of PLP as evidenced by the restoration of complete enzyme activity as well as by the spectral properties. The unique feature of the mechanism proposed for the interaction of OADS with sheep liver SHMT was the formation of PLP as an intermediate.

Animals↗
PubMed2005

Preferred conformers and photochemical (lambda > 200 nm) reactivity of serine and 3,3-dideutero-serine in the neutral form.

A systematic investigation of the conformational potential energy surface of neutral serine [HOCH2CHNH2COOH] and 3,3-dideutero-serine [HOCD2CHNH2COOH] was undertaken, revealing the existence of 61 different minima. The structures and vibrational spectra of the most stable conformers, which were estimated to have relative energies within 7 kJ mol(-1) and account for ca. 93% of the total conformational population at room temperature, were calculated at both the MP2 and DFT/BLYP levels of theory with the 6-311++G(d,p) basis-set and used to interpret the spectroscopic data obtained for the compounds isolated in low-temperature inert matrixes. The assignment of the main spectral infrared features observed in the range 4000-400 cm(-1) to the most stable conformers of serine was undertaken. In addition, UV irradiation (lambda > 200 nm) of the matrix-isolated compounds was also performed, leading to decarboxylation, which was found to be strongly dependent on the conformation assumed by the reactant molecule.

Algorithms↗
PubMed1984

Des-, syn- and anti-oxyimino-delta 3-cephalosporins. Intrinsic reactivity and reaction with RTEM-2 serine beta-lactamase and D-alanyl-D-alanine-cleaving serine and Zn2+-containing peptidases.

The presence and configuration (syn or anti) of an oxyimino group in the 7 (beta)-acyl side chain of 3-cephems do not modify the intrinsic reactivity of the beta-lactam ring, but have highly enzyme-specific effects. When compared with the corresponding desoxyimino beta-lactam compound: (i) with the plasmid-mediated Escherichia coli RTEM-2 serine beta-lactamase, the substrate activity of the anti isomer is increased and that of the syn isomer is decreased; (ii) with the Streptomyces R61 serine D-alanyl-D-alanine cleaving peptidase (a highly penicillin-sensitive enzyme), the rate of enzyme acylation is not or only little affected when the oxyimino group is in the syn configuration, but is decreased when the oxyimino group is in the anti configuration; (iii) with the Actinomadura R39 serine D-alanyl-D-alanine-cleaving peptidase (an exceedingly highly penicillin-sensitive enzyme), the rate of enzyme acylation is unaffected whatever the configuration of the substituent. The oxidation of the sulphur atom of the dihydrothiazine ring on the beta-face of the molecule makes it both a poorer inactivator of the DD-peptidases and a poorer substrate of the beta-lactamase. The Streptomyces albus G Zn2+-containing D-alanyl-D-alanine-cleaving peptidase (a highly penicillin-resistant enzyme) remains highly resistant to all compounds tested.

Carboxypeptidases↗
PubMed1987

Lipopolysaccharide-sensitive serine-protease zymogen (factor C) of horseshoe crab hemocytes. Identification and alignment of proteolytic fragments produced during the activation show that it is a novel type of serine protease.

The horseshoe crab clotting factor, factor C, present in the hemocytes is a serine-protease zymogen activated with lipopolysaccharide. It is a two-chain glycoprotein (Mr = 123,000) composed of a heavy chain (Mr = 80,000) and a light chain (Mr = 43,000) [T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521]. In our continued study of this zymogen, we have now also found a single-chain form of factor C (Mr = 123,000) in the hemocyte lysate. The heavy chain had the NH2-terminal sequence of Ser-Gly-Val-Asp-, consistent with that of the single-chain factor C, indicating that the heavy chain is derived from the NH2-terminal part of the molecule. The light chain had an NH2-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with lipopolysaccharide resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72 amino acid residues) and B chains derived from the light chain were formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar in sequence to a family of repeats in human beta 2-glycoprotein I, complement factors B, protein H, C4b-binding protein, and coagulation factor XIII b subunit. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence-Asp-Ala-Cys-Ser-Gly-Asp-Ser-Gly-Gly-Pro-. These results indicate that horseshoe crab factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine protease by hydrolysis of a specific Phe-Ile peptide bond.

Amino Acid Sequence↗
PubMed1994

Genetics of the serine cycle in Methylobacterium extorquens AM1: cloning, sequence, mutation, and physiological effect of glyA, the gene for serine hydroxymethyltransferase.

The gene (glyA) of Methylobacterium extorquens AM1 encoding serine hydroxymethyltransferase (SHMT), one of the key enzymes of the serine cycle for C1 assimilation, was isolated by using a synthetic oligonucleotide with a sequence based on amino acid sequence conserved in SHMTs from different sources. The amino acid sequence deduced from the gene revealed high similarity to those of known SHMTs. The cloned gene was inactivated by insertion of a kanamycin resistance gene, and recombination of this insertion derivative with the wild-type gene produced an SHMT null mutant. Surprisingly, this mutant had lost its ability to grow on C1 as well as on C2 compounds but was still able to grow on succinate. The DNA fragment containing glyA was shown not to be linked with fragments carrying serine cycle genes identified earlier, making it the fourth chromosomal region of M. extorquens AM1 to be indicated as being involved in C1 assimilation.

Base Sequence↗
PubMed1998

Microtubule-damaging drugs triggered bcl2 phosphorylation-requirement of phosphorylation on both serine-70 and serine-87 residues of bcl2 protein.

Specifically anti-microtubule agents such as taxol, vincristine, vinblastine and dolastatin can trigger Bcl2 phosphorylation at G2-M phase of the cell cycle in malignant cells derived from a variety of human cancers. In this study, the status of Bcl2 phosphorylation was investigated in response to more antimicrotubule agents such as colchicine, colcemid or podophyllotoxin. Although these agents are not currently used for cancer therapy, they were able to trigger Bcl2 phosphorylation with simultaneous apoptosis in cancer cells. Previously, by using extensive site-directed mutagenesis studies we determined that mutation of serine-70 to alanine could not completely abrogate taxol induced Bcl2 phosphorylation. Studies reported here clearly indicate that serine-87 residue along with serine-70 of Bcl2 protein are necessary for microtubule damaging drug induced phosphorylation.

Antineoplastic Agents, Phytogenic↗
PubMed1992

Utilization of an active serine 101----cysteine mutant to demonstrate the proximity of the catalytic serine 101 and histidine 237 residues in thioesterase II.

Thioesterase II is a 29-kDa monomer which, in certain specialized tissues, acts as a chain terminator in fatty acid synthesis by hydrolyzing medium-chain fatty acids from the fatty acid synthase. As with serine proteases, hydrolysis appears to involve acylation of the active site serine residue (Ser-101) assisted by a histidine, tentatively identified as His-237. To determine whether in the folded protein His-237 is close enough to accept a proton from the Ser-101 hydroxyl, we have made use of a Ser101Cys mutant which retains up to 90% of catalytic activity. Unlike the wild-type enzyme, the S101C thioesterase is inhibited with stoichiometric amounts of the bifunctional alkylating reagent 1,3-dibromopropanone. To facilitate identification of the alkylated residue(s), the keto group introduced into the dibromopropanone-modified S101C mutant was radiolabeled by reduction with sodium [3H] borohydride. The protein was then digested and the radiolabeled peptides analyzed by amino acid sequencing and mass spectrometry. The experimental data unambiguously showed that dibromopropanone cross-linked the active site Cys-101 with His-237, demonstrating that these residues are positioned within 5 A of each other. These data strongly support the hypothesis that in the wild-type thioesterase His-237 accepts a proton from Ser-101, thus increasing its nucleophilic character and improving the catalytic efficiency of the enzyme. The possibility that exchange of cysteine and serine active site residues has occurred in the evolution of thioesterases is discussed.

Amino Acid Sequence↗
PubMed1983

The inhibition of the mitogenic stimulation of B lymphocytes by a serine protease inhibitor: commitment to proliferation correlates with an enhanced expression of a cell-associated arginine-specific serine enzyme.

By the use of diisopropylfluorophosphate (DFP) we have been able to show that the mitogenic stimulation of murine B lymphocytes can be maximally inhibited a few hours before commitment of the cells to DNA synthesis. This inhibition was shown to be specific for a serine enzyme(s). The results of experiments designed to determine the location of this enzyme indicated that the mitogens-linked serine enzyme is not a secreted extracellular factor but is cell-associated. Fluorographic analysis of electrophoretic gels of cell homogenates labeled with [3H]DFP revealed the presence of one major and three minor bands which were arginine-specific serine enzymes. In stimulated cells, there was a clear quantitative difference in the amount of [3H]DFP bound to the major band (approx. 23,000 m.w.) suggesting that this protein may be critical to the progression of the cells through the cell cycle into the S phase of DNA synthesis.

Animals↗
PubMed2001

Adsorption of O-Phospho-L-Serine and L-Serine onto Poorly Crystalline Apatite.

The adsorption of phosphoserine and serine was studied to determine the effect of amino acid functional groups on the surface reactivity of synthetic poorly crystalline apatite similar to bone mineral. The experimental results for phosphoserine and serine uptake agree respectively with the Langmuir and Freundlich models. Phosphoserine exhibits stronger adsorption capacity and a higher affinity constant for the surface crystals compared to serine molecules. The enhanced adsorption capacity noted for phosphoserine might be related to the presence of phosphate groups in the molecule, which are specific attachment sites. This observation suggests that the strength of phosphate bonds to the solid surface, especially to calcium ions, is higher than that of carboxyl and hydroxyl ones. Spectroscopic observations provide evidence of an adsorption mechanism involving the anionic species of the amino acids and the surface of the crystals. Thus, a change in the position of the band of carboxyl groups occurred for the adsorbed molecules compared to the native amino acids. This revealed that the molecular residues do interact with apatite surface calcium. The shift noted in the frequencies of the bands associated with carboxylate vibrations is more pronounced for phosphoserine, confirming the stronger interaction noted for this molecule. Based on these results, one can conclude that the sorbent and sorbate charged species play an important role in the mechanism of uptake of the amino acids onto crystal surfaces. This may contribute to a better understanding of the mechanism by which phosphoproteins could influence mineralization processes and caries. Copyright 2001 Academic Press.

Journal Article↗
PubMed1999

Serine 32 and serine 36 of IkappaBalpha are directly phosphorylated by protein kinase CKII in vitro.

IkappaBalpha is an inherently unstable protein which binds to and retains the ubiquitous transcription factor NFkappaB in the cytoplasm of resting cells. A continuous low level translocation of NFkappaB to the nucleus, secondary to the basal turnover of IkappaBalpha, is hypothesized to be necessary for cellular maturation, survival and, potentially, transformation. In response to cellular stimulation by inflammatory cytokines or mitogens, IkappaBalpha is rapidly degraded allowing larger pools of NFkappaB to translocate to the nucleus. Phosphorylation of IkappaBalpha at serine 32 (S32) and serine 36 (S36) is necessary for this stimuli-induced degradation. IKKalpha/beta kinases and p90(rsk1)are involved in stimuli-induced targeting of one or both of these IkappaBalpha sites. Whether other kinases phosphorylate S32 and S36 directly, and if so, what function they serve in NFkappaB activation remains unknown. Here we present evidence of a direct phosphorylation of IkappaBalpha at both S32 and S36 by purified or immunoprecipitated protein kinase CKII (PK-CKII) and a specific in vivo association between IkappaBalpha and PK-CKII. This PK-CKII-specific kinase activity is not found within the IKKalpha/beta-containing signalsome complex and is biochemically distinct from that of the IKKalpha/beta kinases. The identification of an additional N-terminal IkappaBalpha kinase which is constitutively active and not significantly inducible raises numerous possibilities as to its role in cellular function.

2,3-Diphosphoglycerate↗
PubMed1996

A serine alkaline protease from the fungus Conidiobolus coronatus with a distinctly different structure than the serine protease subtilisin Carlsberg.

In view of the functional similarities between subtilisin Carlsberg and the alkaline protease from Conidiobolus coronatus, the biochemical and structural properties of the two enzymes were compared. In spite of their similar biochemical properties, e.g., pH optima, heat stability, molecular mass, pI, esterase activity, and inhibition by diisopropyl fluorophosphate and phenylmethlysulfonylfluoride, the proteases were structurally dissimilar as revealed by (1) their amino acid compositions, (2) their inhibition by subtilisin inhibitor, (3) their immunological response to specific anti-Conidiobolus protease antibody, and (4) their tryptic peptide maps. Our results demonstrate that although they are functionally analogous, the Conidiobolus protease is structurally distinct from subtilisin Carlsberg. The Conidiobolus protease was also different from other bacterial and animal proteases (e.g. pronase, protease K, trypsin, and chymotrypsin) as evidenced by their lack of response to anti-Conidiobolus protease antibody in double diffusion and in neutralization assays. The Conidiobolus serine protease fails to obey the general rule that proteins with similar functions have similar primary sequences and, thus, are evolutionarily related. Our results strengthen the concept of convergent evolution for serine proteases and provide basis for research in evolutionary relationships among fungal, bacterial, and animal proteases.

Amino Acids↗
PubMed1990

Regulatory phosphorylation of serine-15 in maize phosphoenolpyruvate carboxylase by a C4-leaf protein-serine kinase.

We have recently reported that the light-induced changes in the enzymatic and regulatory properties of maize leaf phosphoenolpyruvate carboxylase are attributed to the regulatory seryl phosphorylation of this C4-photosynthesis enzyme. In the present study, the darkform target enzyme was phosphorylated/activated in vitro by a maize leaf protein-serine kinase, and the 32P-labeled regulatory site phosphopeptide was purified from a tryptic digest by metal-ion affinity and reversed-phase chromatography. Automated Edman degradation analysis by covalent protein sequencing technology revealed that the amino acid sequence of this phosphoseryl peptide is His-His-Ser(P)-Ile-Asp-Ala-Gln-Leu-Arg. This nonapeptide, which corresponds exactly to residues 13-21 in the deduced primary sequence of the maize leaf carboxylase, is far removed from recently identified active-site cysteine (Cys-553) and lysine (Lys-606) residues in the C-terminal region of the primary structure. Comparative analysis of the deduced N-terminal sequences of C3-, C4-, and Crassulacean acid metabolism (CAM)-leaf phosphoenolpyruvate carboxylases suggests that the motif of Lys/Arg-X-X-Ser is an important structural requirement of the C4- and CAM-leaf protein-serine kinases.

Amino Acid Sequence↗
PubMed2000

Tau phosphorylation at serine 396 and serine 404 by human recombinant tau protein kinase II inhibits tau's ability to promote microtubule assembly.

In Alzheimer's disease, hyperphosphorylated tau is an integral part of the neurofibrillary tangles that form within neuronal cell bodies and fails to promote microtubule assembly. Dysregulation of the brain-specific tau protein kinase II is reported to play an important role in the pathogenesis of Alzheimer's disease (Patrick, G. N., Zukerberg, L., Nikolic, M., De La Monte, S., Dikkes, P., and Tsai, L.-H. (1999) Nature 402, 615-622). We report here that in vitro phosphorylation of human tau by human recombinant tau protein kinase II severely inhibits the ability of tau to promote microtubule assembly as monitored by tubulin polymerization. The ultrastructure of tau-mediated polymerized tubulin was visualized by electron microscopy and compared with phosphorylated tau. Consistent with the observed slower kinetics of tubulin polymerization, phosphorylated tau is compromised in its ability to generate microtubules. Moreover, we show that phosphorylation of microtubule-associated tau results in tau's dissociation from the microtubules and tubulin depolymerization. Mutational studies with human tau indicate that phosphorylation by tau protein kinase II at serine 396 and serine 404 is primarily responsible for the functional loss of tau-mediated tubulin polymerization. These in vitro results suggest a possible role for tau protein kinase II-mediated tau phosphorylation in initiating the destabilization of microtubules.

Amino Acid Substitution↗