This is the institutional Repository of the Helmholtz Centre for Infection Research in Braunschweig/Germany (HZI), the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken/Germany, the TWINCORE Zentrum für Exprerimentelle und Klinische Infektionsforschung, Hannover/Germany,Helmholtz-Institut für RNA-basierte Infektionsforschung (HIRI), Würzburg/Germany, Braunschweig Integrated Centre for Systems biology (BRICS), Centre for Structural Systems Biology (CSSB) the Study Centre Hannover, Hannover/Germany and the Centre for Individualised Infection Medicine (CiiM).

 

  • PHOSPHOLIPID-GLYCERIDE INTERACTIONS AS REGULATORS OF CARBOXYLESTER LIPASE ADSORPTION AND CATALYSIS

    Brockman, Howard L.; Muderhwa, Jean M.; The Hormel Institute, University of Minnesota, Austin, MN 55912 (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    Lipolysis occurs in the presence of non-substrate, surface-active molecules like phosphatidylcholines (PC’s). The role of PC in the regulation of carboxylester lipase (CEL) was investigated using mixed lipid monolayers. For each substrate an abrupt increase in substrate hydrolysis from <10% to >90%, i.e. switching, occurred at a substratedependent lipid composition. Similar results were obtained for free fatty acid 180 exchange catalyzed by CEL. The lack of hydrolysis at low substrate was not due to an absence of CEL at the interface. Enzyme adsorption to substrate.PC films is consistent with all surface being available, except for 43.5 AZ /molecule of PC. Adsorption of CEL obtained by extrapolation to infinite dilution of PC was 0.92 pmol/cm?, 1/4 of the expected value. CEL adsorption to PCefatty acid films fell short of the values predicted from substrate-containing films. These results suggest that adsorption of CEL to PC-rich films is regulated by clusters or domains of substrate or fatty acid molecules in the PC interface. Such clusters are predicted from stochastic distribution of components but appear to be modulated by lipid-lipid interactions. The stochastic argument further predicts that substrate domains should interconnect if >45% of total surface area is non-excluded by PC. Using data obtained from CEL binding to calculate non-excluded area at each lipid composition, hydrolysis and 180-exchange data are consistent with catalysis being regulated by domain connectivity, i.e. percolation. Being lipid-based, this regulation should occur with other lipases, but the consequences of the organization may be lipase specific.
  • STEREOSELECTIVITY OF LIPASES: Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique

    Ransac, S.; Rogalska, E.; Gargouri, Y.; Deveer, A. M. T. J.; Paltauf, F.; Gancet, C.; Dijkman, R.; De Haas, G. H.; Verger, R.; Centre de Biochimie et de Biologie Moléculaire du C.N.R.S., 31, chemin Joseph Aiguier, 13402 Marseille cedex 9 (France); Laboratoire de Biochimie, Ecole Nationale d'Ingénieurs de Sfax, Route Soukra, 3038 Sfax (Tunisia); Laboratory of biochemistry, C.B.L.E., TransIII, Padualaan 8, 3584 CH, Utrecht (The Netherlands); Institut für Biochemie und Lebensmittelchemie, Technische Universität, A-8010 Graz (Austria); DépartementChimie Fine et Bioconversions, Groupementde Recherches de Lacq, 64170 Artix (France) (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    In the present study, porcine pancreatic lipase (PPL), rabbit gastric lipase (RGL) and humangastric lipase (HGL) stereospecificity towards enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1 ,2)-diacyl-sn-glycerol or enantiomeric 1(3)-alkyl-2-acyl-snglycerol or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the samein both enantiomers, thereby excluding the possibility of effects occuring dueto fatty acid specificity. We observed a PPL sn-3 stereoselectivity when using the enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack ofstereoselectivity observed when using the enantiomeric 1(3)-alkyl- 2,3(1,2)-diacyl-sn-glycerol (triglyceride analogues). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyse the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoalky] diacylpairtested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of RGL, whereas low rates and a clear chiral discrimination was noticed in the case of HGL duringcatalysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers.It is particulary obviousthat in the case of HGL decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylaminoderivatives (diglyceride analogues).
  • THE CRYSTAL STRUCTURE OF LIPASE FROM MUCOR MIEHEI

    Derewenda, Zygmunt; Dodson, Guy; Turkenburg, Johan; Department of Chemistry, University of York, York, UK (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    The crystal structure of lipase from the fungus Mucor miehei has been determined; it has revealed the enzyme's main chain structure as well as the details of the interactions madeby the individual sidechains. The enzymecontains a central 8 strand 6 sheet structure that extends acrossthe full depth of the molecule. Arranged across this, in some of the segmentslinking the strands, are several helices which pack against the sheet structure. There is an N terminal helix which appearsto sit at the centre of the convex surface created by the ß sheet. The serine (144) at the catalytic site has ben identified by chemical experiment. Inspection of the structure at this serine showedit to be part ofa triad: asp... his ... ser, equivalent at the active atomsto that seen in the serine proteases. Thereis no similarity in the lipase main chain structure to those of the trypsin related or the subtilisin related serine proteases - thus the appearanceofthe asp- his - ser triad is an example of an independentsolution of these side chainsfor a catalytic reaction. There is a small helix situated over the catalytic residues, effectively blocking them from the surrounding solvent. This lid explains the inactivity of the enzyme in aqueous conditions. The side chains on this helix are on one side polar and on the other nonpolar. This suggests that underthe influence of the interface at a micelle the lid could be destabilised by non-polar interactions andbe displaced, exposing the catalytic triad to the lipid at the interface.
  • LIPASE KINETICS AT THE TRIACYLGLYCEROL-WATER INTERFACE

    Nury, S.; Gaudry-Rolland, N.; Riviere, C.; Gargouri, Y.; Bois, A.; Lin, M.; Grimaldi, M.; Richou, J.; Verger, R.; Centre de Biochimieet de Biologie Moléculaire du C.N.R.S., 31, chemin Joseph Aiguier, 13402 Marseille cedex 9 (France); LPL,Université de Provence, 3 Place Victor-Hugo, 13331 Marseille Cedex 01 (France); STIL, Université de Toulon, BP 132, 83957 La Garde (France) (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    A new method,-called the "oil-drop method", was developed and adapted to studing the rate of enzymatic reactions, using long chain triacylglycerols, the main physiological substrates of digestive lipases. The method is based on the variations versus time in theoil/waterinterfacial tension (Yo/w) resulting from the accumulation of water insoluble lipolytic products on the surface of a drop (1). Measurements were carried out with pure Porcine Pancreatic Lipase (PPL). This method was also used to detect Human Gastric Lipase (HGL)at low pH,since difficulties were encountered in earlier studies when measuring lipolytic activity under acidic conditions (2). The lipolytic kinetics under high hydrostatic pressure (800 and 1200 bars) were also investigated with the oil-drop method, and a specific two-fold increase in lipase activity was found to have occured. A newprototype is being developed for automatically analyzing the oil-drop profile is being developed in order to improve the data acquisition rate andthe accuracy of the measurements.
  • CRYSTALLOGRAPHIC STUDY OF A RECOMBINANT CUTINASE FROM FUSARIUM SOLANIPISI

    Martinez, Chrislaine; Abergel, Chantal; Cambillau, Christian; de Geus, Pieter; Lauwereys, Mark; Laboratoire de Cristallographie et Cristallisation des Macromolécules Biologiques, Faculté de Médecine Nord, Bvd. P.Dramard, 13326 MARSEILLE CEDEX15 , France; (GBF Gesellschaft für Biotechnologische Forschung mbH, Braunschweig, 1991)
    Cutinases are a group of extracellular fungal hydrolytic enzymes capable of degrading the insoluble lipid polyester matrix,i.e. cutin, which covers the surface of plants. Their weight is 22,000 Da, signifantly lower than all other lipases. A recombinant cutinase from F. solani pisi is expressed and excreted with very high yieldsin E. coli cultures. Cutinase wascrystallized (PEG 6000 15-20% ,pH 7.0 to 10.0,20°C) in space group P21 with cell dimensions 35.1A,67.4A,37.05 A, ß=94°.They diffract to 1.5 A resolution (Rsym=4.41%). Data from native and derivatives have been collected. MIR phasingis in progress.

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