The present study aimed at examining the physiological characteristics and metabolic demands of badminton single match play. Twelve internationally ranked badminton players (eight women and four men) performed an incremental treadmill test [VO(2peak = )50.3 +/- 4.1 ml min(-1) kg(-1) (women) and 61.8 +/- 5.9 ml min(-1) kg(-1) (men), respectively]. On a separate day, they played a simulated badminton match of two 15 min with simultaneous gas exchange (breath-by-breath) and heart rate measurements. Additionally, blood lactate concentrations were determined before, after 15 min and at the end of the match. Furthermore, the duration of rallies and rests in between, the score as well as the number of shots per rally were recorded. A total of 630 rallies was analysed. Mean rally and rest duration were 5.5 +/- 4.4 s and 11.4 +/- 6.0 s, respectively, with an average 5.1 +/- 3.9 shots played per rally. Mean oxygen uptake (VO(2)), heart rate (HR), and blood lactate concentrations during badminton matches were 39.6 +/- 5.7 ml min(-1) kg(-1) (73.3% VO(2peak)), 169 +/- 9 min(-1) (89.0% HR(peak)) and 1.9 +/- 0.7 mmol l(-1), respectively. For a single subject 95% confidence intervals for VO(2) and HR during match play were on average 45.7-100.9% VO(2peak) and 78.3-99.8% HR(peak). High average intensity of badminton match play and considerable variability of several physiological variables demonstrate the importance of anaerobic alactacid and aerobic energy production in competitive badminton. A well-developed aerobic endurance capacity seems necessary for fast recovery between rallies or intensive training workouts.

We provide a mechanism for the activity of pectin methylesterase (PME), the enzyme that catalyses the essential first step in bacterial invasion of plant tissues. The complexes formed in the crystal using specifically methylated pectins, together with kinetic measurements of directed mutants, provide clear insights at atomic resolution into the specificity and the processive action of the Erwinia chrysanthemi enzyme. Product complexes provide additional snapshots along the reaction coordinate. We previously revealed that PME is a novel aspartic-esterase possessing parallel b-helix architecture and now show that the two conserved aspartates are the nucleophile and general acid-base in the mechanism, respectively. Other conserved residues at the catalytic centre are shown to be essential for substrate binding or transition state stabilisation. The preferential binding of methylated sugar residues upstream of the catalytic site, and demethylated residues downstream, drives the enzyme along the pectin molecule and accounts for the sequential pattern of demethylation produced by both bacterial and plant PMEs.

Pectate lyases harness anti beta-elimination chemistry to cleave the alpha-1,4 linkage in the homogalacturonan region of plant cell wall pectin. We have studied the binding of five pectic oligosaccharides to Bacillus subtilis pectate lyase in crystals of the inactive enzyme in which the catalytic base is substituted with alanine (R279A). We discover that the three central subsites (-1, +1, and +2) have a profound preference for galacturonate but that the distal subsites can accommodate methylated galacturonate. It is reasonable to assume therefore that pectate lyase can cleave pectin with three consecutive galacturonate residues. The enzyme in the absence of substrate binds a single calcium ion, and we show that two additional calcium ions bind between enzyme and substrate carboxylates occupying the +1 subsite in the Michaelis complex. The substrate binds less intimately to the enzyme in a complex made with a catalytic base in place but in the absence of the calcium ions and an adjacent lysine. In this complex, the catalytic base is correctly positioned to abstract the C5 proton, but there are no calcium ions binding the carboxylate at the +1 subsite. It is clear, therefore, that the catalytic calcium ions and adjacent lysine promote catalysis by acidifying the alpha-proton, facilitating its abstraction by the base. There is also clear evidence that binding distorts the relaxed 2(1) or 3(1) helical conformation of the oligosaccharides in the region of the scissile bond.

SummaryThe type II secretion system (T2SS) is widely exploited by proteobacteria to secrete enzymes and toxins involved in bacterial survival and pathogenesis. The outer membrane pore formed by the secretin OutD and the inner membrane protein OutC are two key components of the secretion complex, involved in secretion specificity. Here, we show that the periplasmic regions of OutC and OutD interact directly and map the interaction site of OutC to a 20-residue peptide named OutCsip (secretin interacting peptide, residues 139-158). This peptide interacts in vitro with two distinct sites of the periplasmic region of OutD, one located on the N0 subdomain and another overlapping the N2-N3Ј subdomains. The two interaction sites of OutD have different modes of binding to OutCsip. A single substitution, V143S, located within OutCsip prevents its interaction with one of the two binding sites of OutD and fully inactivates the T2SS. We show that the N0 subdomain of OutD interacts also with a second binding site within OutC located in the region proximal to the transmembrane segment. We suggest that successive interactions between these distinct regions of OutC and OutD may have functional importance in switching the secretion machine.

Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full-length rat and human dihydroorotate dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 micromol x min(-1) x mg(-1) at pH optimum of 8.0--8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6--1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K(m) = 11 microM dihydroorotate; K(m) = 7 microM ubiquinone) and human enzyme (K(m) = 10 microM dihydroorotate; K(m) = 14 microM ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K(m) = 26 microM dihydroorotate and K(m) = 62 microM ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the 'slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.

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Background: Tissue factor pathway inhibitor (TFPI) inhibits coagulation factors Xa and VIIa. Results: A de novo synthesized 20-mer peptide that binds to TFPI was structurally and functionally characterized. Conclusion:The peptide binds to the Kunitz domain 1 of TFPI and blocks inhibition of factor Xa and factor VIIa by TFPI. Significance: The peptide can potentially prevent bleeding in hemophilia patients.

Limited proteolysis of the pyruvate decarboxylase (E1, alpha2beta2) component of the pyruvate dehydrogenase (PDH) multienzyme complex of Bacillus stearothermophilus has indicated the importance for catalysis of a site (Tyr281-Arg282) in the E1alpha subunit (Chauhan, H.J., Domingo, G.J., Jung, H.-I. & Perham, R.N. (2000) Eur. J. Biochem. 267, 7158-7169). This site appears to be conserved in the alpha-subunit of heterotetrameric E1s and multiple sequence alignments suggest that there are additional conserved amino-acid residues in this region, part of a common pattern with the consensus sequence -YR-H-D-YR-DE-. This region lies about 50 amino acids on the C-terminal side of a 30-residue motif previously recognized as involved in binding thiamin diphosphate (ThDP) in all ThDP-dependent enzymes. The role of individual residues in this set of conserved amino acids in the E1alpha chain was investigated by means of site-directed mutagenesis. We propose that particular residues are involved in: (a) binding the 2-oxo acid substrate, (b) decarboxylation of the 2-oxo acid and reductive acetylation of the tethered lipoyl domain in the PDH complex, (c) an "open-close" mechanism of the active site, and (d) phosphorylation by the E1-specific kinase (in eukaryotic PDH and branched chain 2-oxo acid dehydrogenase complexes).