Bacterial Cell Wall Hydrolysis by Lysozyme

1
Bacterial Cell Wall Hydrolysis by Lysozyme

• We have examined the structure of the
  well-characterized hen egg white (HEW) lysozyme
  in considerable detail. This enzyme catalyzes
  the hydrolysis of bacterial wall polysaccharides
  of the following structure
• The alternating monomers of this polysaccharide
  are N-acetylmuramic acid (NAM) and
  N-acetylglucosamine (NAG)
• Hydrolysis occurs selectively at the noted
  positions, with no cleavage of the polysaccharide
  at C-1 of the NAG residues.
• This is a mild technique for cell disruption
  that, due to the cost of lysozyme, is rarely used
  for industrial enzyme isolation processes.

2
Characterization of the Active Site

• While the mechanism of every catalytic process is
  very difficult to determine, enzyme mediated
  reactions are particularly troublesome, as we
  have very little knowledge of which functional
  groups participate directly in the catalysis.
• Kinetic data is useful for design purposes, but
  it rarely leads to a reliable reaction mechanism
  without supporting information.
• Product distribution analysis of model compounds
  that approximate the reactive functionality of
  the polysaccharide.
• Crystallographic analysis of the enzyme and
  various enzyme-inhibitor or enzyme-substrate
  complexes
• Assessment of the influence of select amino acid
  substituent modification
• Measurement of kinetic isotope effects
• All four of these techniques have been applied to
  lysozyme catalysis, and we will examine the first
  three to better understand the nature of
  enzyme-substrate interactions.

3
Lysozyme Activity Studies - Model Compounds

• The cleavage patterns for acetylglucosamine
  oligomers
• are not consistent with a random attack of the
  enzyme.
• Cleavage occurs at an appreciable rate only for
  hexamers or higher oligomers and occurs between
  mers 4 and 5.
• Indicates that a hexasaccharide is incorporated
  by the catalytic site of the enzyme, and a unique
  mode of substrate activation must establish a
  preferred hydrolysis pathway.

4
Crystallographic Studies of Lysozyme-Inhibitor
Complexes

• Once the crystal structure of an enzyme has been
  determined the structures of isomorphous crystals
  that contain additional molecules may be
  determined without difficulty.
• This method has been used to explore the
  interactions between lysozyme and a wide range of
  substrate-related inhibitor molecules.

Perspective drawing of the main chain
conformation of lysozyme Elevation from
active-site side of the molecule. Only a
positions of a-carbon atoms are shown.
5
Crystallographic Studies of Lysozyme-Inhibitor
Complexes

• The trisaccharide of NAG forms a relatively
  stable complex with lysozyme that has been
  characterized by crystallography. The position
  of the three NAG groups is illustrated at the
  top-right of the structure.
• The other three groups have been
• positioned by molecular
• modeling, as they cannot be
• isolated.
• N-acetylglucosamine (NAG) alone
• binds to the enzyme with H-bonds between the NH
  and carbonyl
• oxygens of its acetamido side chain and the main
  peptide chain and CO and NH groups of residues
  107 and 59.

6
Active Site Determination Chemical Modification

• If the chemical modification of a particular
  amino acid side chain results in enzyme
  deactivation, then the residue in question is
  located at the active site, provided that the
  modification can be prevented by the presence of
  excess substrate or inhibitor. The following is
  a summary of specific modifications used to
  determine the activity of individual residues
• A. Amino (Lysyl e-amino, 1,13, 33, 96, 97,116)
• All 6 lysine residues are on the surface of the
  enzyme, with Lys 33 situated in the very bottom
  of the cleft.
• Little effect of modifications of this type on
  lytic activity is observed, suggesting that these
  amino acid residues do not participate directly
  in catalysis.

7
Active Site Determination Chemical Modification

• B. Arginine (5, 14, 21, 45, 61, 68, 73, 112, 114,
  125, 128)
• All but one Arg is located on the surface of the
  enzyme, but Arg 114 is believed to form two
  hydrogen bonds with the saccharide.
• Modification of 7 of the 11 Arg residues had
  little influence on the activity of lysozyme on
  NAG4.
• C. Glutamic Acid 7, 35 Aspartic Acid 18, 48,
  52, 66, 87, 101, 111
• Modification studies of carboxyl groups have
  provided unequivocal evidence for the involvement
  of these residues in catalysis.
• Exhaustive esterification of lysozyme with acid
  alcohol results in a loss of enzyme activity

8
Active Site Determination Chemical Modification

• C. Carboxyl Groups Continued
• Two amino acid residues in particular (Asp 52 and
  Glu 35) have been implicated in several
  modification studies.
• Modification of Asp 52 was inhibited by the
  presence of substrate
• Selective oxidation of Glu 35 with iodine
  denatures the enzyme.
• D. Cysteine (6-127, 30-115, 64-80, 76-94)
• Reduction of disulfide crosslinks denatures the
  enzyme, although 6-127 can be opened without
  deactivation. This is reversible, as air
  oxidation regenerates enzymatic activity with
  high yield.
• E. Histidine (15)
• Alkylation of the single histidine has been shown
  to have little influence on lytic activity.

9
Active Site Determination Chemical Modification

• F. Methionine (12,105)
• Cyanogen bromide in 70 formic acid cleaves both
  methionyl peptide bonds without modification of
  other amino acid sequences.
• This reduces activity to 10 of the native
  enzyme, despite the fact that both residues are
  buried within the enzyme structure and
  participate through non-polar contacts with other
  residues.
• G. Tryptophan (28, 62, 63, 108, 111, 123)
• Three of six tryptophans are believed to be
  positioned in the active site. Oxidation with
  N-bromosuccinimide inactivates the enzyme.
• Selective oxidation of Trp 108
• with iodine is blocked by
• substrate. Trp is also in close
• proximity to Glu 35.

10
Binding of Lysozyme to Hexa-N-Acetylglucosamine

• Schematic illustration of the active site in the
  cleft region of lysozyme. A through F represent
  the glycosyl moieties of a
• hexa-saccharide. Some of the amino
• acids in the cleft region near
• these subsites of the
• active site
• are shown.

11
Model of the HEW Lysozyme Site

• Schematic diagram showing the specificity of
  lysozyme for hexa-saccharide substrates.
• Six subsites A-F on the enzyme bind the sugar
  residues. Alternate sites interact with the
  acetamido side chains (a), and these sites are
  unable to accommodate MurNAe residues with their
  lactyl side chains (P).
• Site D cannot bind a sugar residue without
  distortion, and the glycosidic linkage that is
  cleaved binds between sites D and E as shown by
  the arrow.

12
Proposed Catalytic Mechanism

• 1. The saccharide binds in the enzyme cleft with
  residue D distorted to a conformation resembling
  the half-chair.
• 2. Bond rearrangement to yield a carbenium ion
  proceeds at a rate enhanced through several
  contributions
• A. Glu 35 acts as a general acid catalyst,
  donating H to the glycosidic oxygen.
• B. Asp 52 bears a negative charge that favors
  formation of the carbenium ion.
• C. The ring conformation is close to that
  required in the transition state.
• D. The nonpolar nature of the cleft possibly
  enhances reaction rate.
• 3. The enzyme-bound carbonium ion is stabilized
  by neighboring charges of Asp 52 and Glu 35, the
  latter having deprotonated in bond rearrangement.
  
• 4. The aglycone diffuses away, and reaction with
  water or another acceptor completes the process.

13
pH Dependence of Enzyme Activity

• Since the characteristics of ionizable side
  chains of amino acids depend on pH, enzyme
  activity varies with pH shifts.
• At extremes of pH, the tertiary structure of the
  protein may be disrupted and the enzyme
  denatured.
• Even at moderate pH values where tertiary
  structure is unaffected, enzyme activity may
  depend on the degree of ionization of certain
  amino acid side chains
• pH can therefore affect enzyme
• conformation, substrate binding
• and the ability of active side
• groups to participate in catalysis,
• as shown here for three
• representative enzymes.

14
Survey of Ionizable Enzyme Groups

• The ionizable groups which contribute to the
  acid-base properties of proteins, shown with
  their approximate pKa values. These can vary by
  several pH units depending on their environment
  in the protein.

15
pH Dependence of Enzyme Activity

• Recall our discussion of lysozyme, where Asp52
  was believed to exist in its conjugate base
  (RCOO-) form, while Glu35 is thought to be active
  in its acidic state (RCOOH).
• pH will dictate the degree of protonation of
  these residues, creating an optimum that is
  dependent on their pKas.
• Graphs of Vmax against pH, at constant E, where
  catalytic activity depends on the simultaneous
  presence of EY- and EZH
• (a) where pKy and pKz are more than 2 units apart
• (b) where pKy and pKz are less than 2 units apart

16
pH Dependence of Enzyme Activity

• Consider a reaction that requires the conjugate
  acid/base pair HA/A- to be in its basic form, and
  the conjugate acid/base pair HB/B- to be in its
  acidic state. The system speciation will change
  with pH, as follows.