Summary of the Investigation of the Mechanism of Catalysis of Lysozyme



Lysozyme is an enzyme that has the ability to catalyze the hydrolysis of complex polysaccharides that form bacterial cell walls. The protein has 129 amino acid residues, a MW of 14,600 D, and four -S-S- bonds. The natural substrate is a linear polymer of alternate residues of N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM). This last is N-acetyl glucosamine with a lactyl substitution in carbon 3. The connection between these residues is by (14) glycosidic linkages. An important piece of information for the elucidation of the mechanism of catalysis is that the links whose hydrolysis is catalyzed by the enzyme are the NAM-NAG bonds, not the NAG-NAM ones. In addition to this polymer, the enzyme also hydrolyzed polymers of NAG found in the chitinous shell of crustacea.

During the mid1960's, Phillips et al. conducted a series of studies on hen egg white lysozyme that led to the clarification of the mechanism of action of the enzyme. Their first contribution was the X-ray diffraction studies on crystals of the enzyme with enough resolution to pinpoint the position in space of most of its atoms with an error of not more than about 2 Angstrom units. Construction of physical models of the molecule based on these data showed the presence of a large cleft on its surface. (NAG)3, the NAG trimer, was known to be a powerful competitive inhibitor of the enzyme, which means that it binds to the same site on the enzyme as the substrate. In order to find where on the enzyme did (NAG)3 bind, this trimer was added to enzyme crystals in their mother liquid and allowed to diffuse into the crystals. Then, these crystals were also studied with X-ray diffraction and the comparison between the enzyme model with and without (NAG)3 showed that the inhibitor bound to the cleft, but occupying only half of its length. This showed that the cleft was at least part of the substrate's binding site. Playing around with physical models of the molecules of enzyme and of NAG-NAM polymers resulted in some important findings:

  1. The cleft is completely filled by six sugar residues
  2. The weak interactions (hydrogen bonds, etc,) that probably determine the substrate binding and the amino acid residues involved in them could be identified
  3. Starting from one end of the cleft chosen arbitrarily, the sites where six consecutive sugar residues bind were numbered. It was found that binding of NAM to the third site was not possible because the bulkiness of the lactyl group could not be made to fit in it. NAG, on the other hand, has no difficulty fitting in it.

This determines that site 3 must be filled with NAG and the only way in which the natural substrate can bind is

 

                                 1          2           3          4          5           6

.... NAG - NAM - NAG - NAM - NAG - NAM - NAG - NAM - NAG - NAM...

 

Since the glycosidic bond that is hydrolyzed is the NAM - NAG bond, this hydrolysis must take place between sites 2 -3 or 4 -5. In addition, by far the most abundant products of the hydrolysis of (NAG)6 are (NAG)4 and (NAG)2.

The evidence against the catalytic site being in 2 -3 is compelling because (NAG)3 binds to

1 - 2 - 3 thereby preventing the binding of longer polymers, but the rate at which it is hydrolyzed is negligible. Consequently, 4 - 5 emerges as the only reasonable candidate. In addition, the standard free energy of binding of the sugar residues to the six sites and the rates of hydrolysis obtained with polymers of different lengths provide supportive information:

Site                             1                2              3             4           5             6

 

)G0 of binding           - 8              -13          -20         +16        -10         -12

 (KJ/mole)  

Number of NAG            

residues in polymer             3            4                 5                      6                       7

 

Relative rate of

hydrolysis                            1            8            4000                30000                30000

  1. The +16 KJ/mole of free energy of binding to site 4 indicates a possible distortion of the substrate to force it to fit the site. In other words, the negative free energy of binding to the other sites is partially used to force a distortion of the fourth residue so that it fits. Observations of physical models of the molecules show that this distortion consists of a flattening of one end of the chair configuration of the sugar ring. Furthermore, a comparison of the binding constants of (NAG)4 and of its lactone (whose molecule shows that flatten shape) shows that the binding of the lactone is 3600 greater.
  2. The rate of hydrolysis of the NAG trimer is almost unmeasurable. Still, it has been assigned an arbitrary number of 1. The tetramer also hydrolyzes very slowly, but a bit faster probably because of the greater probability of its binding such that its molecule spans the active (catalytic) site of the enzyme. The 500-fold increase from tetramer to pentamer stems from that same effect aided by the contribution to the free energy of binding of that fifth residue. This reinforces the notion that the 4 - 5 connection is the one being split. The hexamer has one more residue to contribute to binding strength, while longer polymers do not change this situation because the extra residues do not fit in the binding site.

In order to look for groups in the enzyme probably involved in the catalysis, it is necessary to determine which of the two bonds of the oxygen in the glycosidic link is hydrolyzed. This was accomplished by hydrolyzing (NAG)6 in the presence of H218O and finding that the heavy oxygen became attached to (NAG)4 instead of to the dimer. This pointed to a bond with two acidic residues close by: Glu 35 and Asp 52. In order to determine whether these residues are involved in catalysis, the enzyme was treated by Koshland's method to modify available carboxylic groups. When the enzyme was treated in the absence of substrate, the result was an inactive enzyme with all the carboxylic groups modified. If the treatment was applied to the enzyme in the presence of substrate, Asp 52 and Glu 35 were not modified and the enzyme did not lose activity. The pKa's of Glu and Asp and their environments in the enzyme are such that, at the optimum pH for the enzyme (5.0), Glu 35 will be protonated while Asp 52 will not. This finding, together with the positive G of binding and the negative G of binding of the lactone at position 4, suggest a mechanism of catalysis.

The protonated carboxylic group of Glu 35 attacks the glycosidic linkage on the side of residue 4, thereby leaving a positive charge on carbon 1 of that residue. This carbonium ion is partially stabilized by the negative charge in the adjacent carboxylic group of Asp 52 and imparts a partially flattened conformation to its ring similar to that seen in the lactones. The carbonium ion form is an unstable transition state that fulfills the requirement of binding to the enzyme with greater strength than the substrate, since it has the shape that fits site 4 without strain. Surrounding water will then supply an H+ to reprotonate Glu 35 and an OH- to link to the positive carbon and allow residue 4 to return to the chair configuration. The conversion of the substrate into the carbonium ion transition state, which would require a large energy of activation, can be considered as being facilitated by the contribution of the negative free energy of binding of the substrate to the distortion of the shape of residue 4 to fit its binding site on the enzyme and by the stabilization of the carbonium's positive charge by the proximity of the negative aspartate side chain.


Some additional evidence that has contributed to the development of this model of lysozyme function have been withheld from this presentation in the interest of brevity and clarity.


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