Lactase

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publicaffairs.uth.tmc.edu/.../got_milk.jpg
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www.manekineko.us
www.epinions.com
www.goodnessdirect.co.uk
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Lactose Intolerance

• The products shown on the previous slide are
  various brands of a food additive used by people
  who are lactose intolerant.
• What type of products can such people not
  consume?
• What is missing in the digestive system of people
  who cant digest lactose?

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Lactose Intolerance LACTASE deficiency
www.ecofriend.org

• Lactase is the enzyme that breaks down the milk
  sugar lactose.

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Reaction Catalyzed by Lactase
(ensiweb/ lessons/tp.milk3.html)
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This reaction is a hydrolysis

• A hydrolysis is a reaction in which a water
  molecule reacts with a compound, after the bond
  between two parts of the molecule has been
  weakened or broken by the activity of an enzyme.

(http//www.elmhurst.edu/chm/vchembook/546lactose
.html)
http//ghs.gresham.k12.or.us/science/ps/sci/soph/o
rganic/dehydration.htm
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b-Galactosidase

• Because the products of the hydrolysis reaction
  include galactose, lactase is called
  b-Galactoside enzyme
• It is present mainly along the brush-border
  membrane of the entrocytes lining the villi of
  the small intestine

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Protein Structure of Lactase

• Lactase is a tetramer, composed of two A chains
  and two B chains. It is part of the Family 1
  glycohydrolases.

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Primary Protein Structure amino acid sequence

• 1 melswhvvfi allsfscwgs dwesdrnfis tagpltndll
  hnlsgllgdq ssnfvagdkd
• 61 myvchqplpt flpeyfsslh asqithykvf
  lswaqllpag stqnpdektv qcyrrllkal
• 121 ktarlqpmvi lhhqtlpast lrrteafadl
  fadyatfafh sfgdlvgiwf tfsdleevik
• 181 elphqesras qlqtlsdahr kayeiyhesy
  afqggklsvv lraedipell leppisalaq
• 241 dtvdflsldl syecqneasl rqklsklqti
  epkvkvfifn lklpdcpstm knpasllfsl
• 301 feainkdqvl tigfdinefl scsssskksm
  scsltgslal qpdqqqdhet tdsspasayq
• 361 rvweafanqs raerdaflqd tfpegflwga
  stgafnvegg waeggrgvsi wdprrplntt
• 421 egqatlevas dsyhkvasdv allcglraqv
  ykfsiswsri fpmghgssps lpgvayynkl
• 481 idrlqdagie pmatlfhwdl pqalqdhggw
  qnesvvdafl dyaafcfstf gdrvklwvtf
• 541 hepwvmsyag ygtgqhppgi sdpgvasfkv
  ahlvlkahar twhhynshhr pqqqghvgiv
• 601 lnsdwaepls perpedlras erflhfmlgw
  fahpvfvdgd ypatlrtqiq qmnrqcshpv
• 661 aqlpefteae kqllkgsadf lglshytsrl
  isnapqntci psydtiggfs qhvnhvwpqt
• 721 ssswirvvpw girrllqfvs leytrgkvpi
  ylagngmpig esenlfddsl rvdyfnqyin
• 781 evlkaikeds vdvrsyiars lidgfegpsg
  ysqrfglhhv nfsdssksrt prksayffts
• 841 iiekngfltk gakrllppnt vnlpskvraf
  tfpsevpska kvvwekfssq pkferdlfyh
• 901 gtfrddflwg vsssayqieg awdadgkgps
  iwdnfthtpg snvkdnatgd iacdsyhqld
• 961 adlnmlralk vkayrfsisw srifptgrns
  sinshgvdyy nrlinglvas nifpmvtlfh
• 1021 wdlpqalqdi ggwenpalid lfdsyadfcf
  qtfgdrvkfw mtfnepmyla wlgygsgefp
• 1081 pgvkdpgwap yriahavika harvyhtyde
  kyrqeqkgvi slslsthwae pkspgvprdv

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Representations of Secondary Structure

• The secondary structure of lactase is
• A chain
• B chain
• There are several ways to graphically represent
• the secondary structure

(Protein Data Bank, 2007)
(Protein Data Bank, 2007)
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Secondary Structure
A Chain
B Chain
(Protein Data Bank, 2007)
(Protein Data Bank, 2007)
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Protein X-ray Structure
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Tertiary Structure

• Crystallographic studies of family 1 glycosidases
  indicate that it has a classic (b/a)8 barrel fold
  
• The active site of b-glycosidases is located in
  the C-terminal portion of the b-barrel and is
  surrounded by loops connecting the a-helix to the
  b-barrel strands.2

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Topology Diagram of Ssb-Gly1

• All b-glucosidases from family 1 share similarity
  in tertiary structure, a (ba)8 barrel
• The a helices, shown as cylinders, and b-strands,
  shown as arrows, of the (ba)8 barrel are colored
  red and green, respectively.

(1997, Aguilar, et al.)
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Quaternary Structure

• Structural analysis reveals a large proportion of
  surface ion-pairs involved in networks that cross
  link sequentially separate structures on the
  protein surface.
• There are an unusually large number of solvent
  molecules buried in the hydrophobic cavities
  between the sequentially separate structures in
  the protein core.1

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Active Site

• Family 1 glycohydrolases share the following
  feature a central indentation at one end of the
  radial channel that contains a high concentration
  of residues, one of which, glutamic acid, has
  been reported as the general acid responsible for
  protonating the glycosidic oxygen.
• This residue is found within a tunnel, which is
  long enough to accommodate up to five linked
  sugar units.1

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Glutamic Acid Carboxylic Acid Groups

• The mechanism involves two active site carboxylic
  acid groups, one functioning as the nucleophile,
  attacking at the sugar anomeric center to form
  the glycosyl-enzyme intermediate.
• The other carboxylic acid group acts as an
  acid/base catalyst, protonating the glycosidic
  oxygen in the first step and deprotonating the
  water in the second step.

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Ligands

• The b-glycosidases active site is divided into
  several subsites large enough to bind a
  monosaccharide unit. Within each subsite the side
  of a non-polar amino acid residue forms a
  platform, which is a support for the ligand.
  Polar amino acid residues may be found
  distributed around the ligand and form H-bonds
  with it.2
• Graphics from PDB Ligand Program

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Ligands

• Sodium and Magnesium Atoms also act as binding
  sites

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Mechanism3

• The hydrolysis is a two step mechanism
• Formation of a glycosyl-enzyme with
• concurrent aglycone departure
• Hydrolysis of the glycosyl-enzyme by a water
  molecule
• Glycosilation is believed to involve a
• nucleophilic attack by a glutamic acid residue.
• The water molecule that mediates deglycosilation
• may be activated by a second Glu acting as a
  base, or may
• initiate its own nucleophilic attack without base
  activation.

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Mechanism4
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Lactase Activity

• While newborn mammals are dependent on high
  levels of lactase for hydrolysis of the lactose
  in milk, most lose lactase activity after weaning
• In humans, two phenotypes exist
• lactose intolerance, called hypolactasia
• lactose tolerance, called lactase persistence
• Lactose persistence is most common among people
  of Northern European descent.5

(1992, Rivera-Sagredo)
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Enzyme Kinetics

• When data are generated for the initial substrate
  concentration and the initial rate of the
  reaction and the results plotted as a graph of
  initial rate vs. substrate concentration many
  enzymes exhibit a rectangular hyperbolic curve
  like the one shown in the diagram.

(http//www.le.ac.uk/by/teach/biochemweb/tutorials
/michment2print.html)
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Michaelis-Menten Equation
If the substrate, A, reacts enzyme E, and turns
into B with rate constant k-1 and then proceeds
to C, regenerating enzyme E, the mechanism can be
described using the Michaelis-Menten equation
Where KM is the Michaelis constant.
(http//www.ch.cam.ac.uk/magnus/michmenten.html)
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Catalytic Parameters

• The specific activity of lactose is 14.5
  mmol/minmg
• Glycoside hydrolases have been shown to
  accelerate the rate of hydrolysis by a factor of
  1017. Km values for this family are around 10-5M,
  and the dissociation constant of the
  enzyme-substrate complex is about 10-22. At a pH
  of 6.5, the kcat was determined to be 4.2 sec-1. 5

(1992, Rivera-Sagredo, et al.)
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Enzyme Regulation

• 33 of the worlds adult population are affected
  by lactose intolerance, and it is the most
  frequent genetically based syndrome in man.5
• It is regulated at the transcriptional level
  (mRNA) as well as by post transcriptional
  mechanisms modulate its final expression.6

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Hydropathy Plot7
Strong negative peaks indicate possible surface
regions of globular proteins
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What does it mean?

• Each amino acid is given a score between 4.6 and
  -4.6 that tells its hydrophobicity (its fear of
  water
• 4.6 is the most hydrophobic and -4.6 is the most
  hydrophilic (water loving
• These scores are graphed for all of the amino
  acids in the peptide
• These graphs can be used to determine globular
  and transmembrane proteins
• The Gravy score is the average hydropathy for
  all the proteins in the structure
• Globular proteins have lower Gravy scores than
  transmembrane proteins6
• (http//gcat.davidson.edu/rakarnik/KD.html)

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Lactase

• Is lactase a globular protein?
• Where in the graph are regions that show
  particular hydrophilicity? Hydrophobicity?

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Specificity

• b-Glycosidases are active on a large range of
  substrates. The molecular basis for the
  specificity is a hydrogen bond network involving
  at least 5 active site amino acid residues and 4
  substrate hydroxyls.
• Studies on Ss-b-Gly indicate that the catalytic
  efficiency of the enzyme increases as the
  substrate length increases.
• The hydrogen bonding
• network may determine
• the glycone specificity.

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Sources

• 1. Aguilar, C. F. Sanderson, I. Moracci, M.
  Ciaramella, M. Nucci, R. Rossi, M. Pearl, L.
  H., Crystal structure of the beta-glycosidase
  from the hyperthermophilic archeon Sulfolobus
  solfataricus resilience as a key factor in
  thermostability. Journal of Molecular Biology
  1997, 271, (5), 789-802.
• Marana, S. R., Molecular basis of substrate
  specificity in family 1 glycoside hydrolases.
  IUBMB Life 2006, 58, (2), 63-73.
• Mian, I. S., Sequence, Structural, Functional,
  and Phylogenetic Analyses of Three Glycosidase
  Families. Blood Cells, Molecules, and Diseases
  1998, 24, (2), 83-100.
• Ly, H. D. Withers, S. G., MUTAGENESIS OF
  GLYCOSIDASES. Annual Review of Biochemistry 1999,
  68, (1), 487-522.
• Rivera-Sagredo, A. Cañada, F. J. Nieto, O.
  Jimenez-Barbero, J. Martin-Lomas, M., Substrate
  specificity of small-intestinal lactase. European
  Journal of Biochemistry 1992, 209, (1), 415-422.
• J T Troelsen, C Mitchelmore, N Spodsberg, A M
  Jensen, O Norén, and H Sjöström. Biochem J. 1997
  March 15 322(Pt 3) 833838.
• Jonhson, S. e. a. A One Stop Site for Kyte
  Doolittle Hydropathy Plots. (July 26),
  http//gcat.davidson.edu/rakarnik/KD.html.
• Fourage, L. Dion, M. Colas, B., Kinetic study
  of a thermostable ß-glycosidase of Thermus
  thermophilus. Effects of temperature and glucose
  on hydrolysis and transglycosylation reactions.
  Glycoconjugate Journal 2000, 17, (6), 377-383.