LYSOZYME: Mechanism Today I want to start our discussion of the mechanisms of enzymes. We'll start with a brief look at lysozyme and then start on the serine proteases. This enzyme typifies glycoside hydrolases in many ways, and has played an important historical role in the development of our understanding of enzyme mechanisms.

Lysozyme has an unusual history. Its 3-D structure was first reported by Phillips and coworkers in 1965. The crystallographic structure allowed Phillips et al. to propose a mechanism, which has stood the test of time rather well. It is interesting that not only was lysozyme (HEW) the first enzyme whose structure was determined by X-ray diffraction, but at that time virtually nothing was known about its catalytic mechanism (in contrast to chymotrypsin (whose structure was the next solved), for example). This is due in great part to technical difficulties in studying the kinetics of lysozyme catalysis.

The reaction catalyzed is the hydrolysis of bacterial cell-wall polysaccharide (peptidoglycan), (NAG-NAM)n, or of chitin, which is a polymer of NAG (e. g. insect exoskeleton). Typical bacterial cell-wall looks like:

The enzyme is an endo-glycosidase, hydrolyzing adjacent to the N-acetylmuramic acid moieties. Although the natural substrate is NAG-NAM-NAG-NAM- ..., it is also very active toward chitin (the exoskeleton of crustacea), NAG-NAG-NAG-NAG-.... .

Hydrolysis occurs between NAM-NAG. The best small substrates are the NAG-NAM trimer (i. e. six sugars) and chitohexose.

Lysozyme Structure:

Lysozyme has a cleft running across its waist, in which are found 6 sugar binding subsites ( A-F).

A number of studies have indicated that the structure in the crystalline state is very similar to that in solution. These include the fact that different crystal forms of the enzyme give the same structure, as do crystals grown form different media, the crystals are approx. 50% solvent, so there are relatively few inter-molecular contacts in the crystal, the crystals (for tortoise lysozyme) are catalytically active, and NMR studies of the enzyme in solution show it to be consistent with the crystal structure. Several proteins have had their structures determined both by NMR in solution and X-ray crystallography: in general, excellent agreement is found throughout the molecule.

Proposed mechanism of action for lysozyme:

There are 3 main methods by which enzyme mechanism information is obtained:

Chemical modification and site-directed mutagenesis studies (to identify the reactive side-chains) Kinetics studies, including the use of inhibitors

Structural studies, including X-ray diffraction (often also of inhibitor complexes)

In addition theoretical calculations are sometimes useful.

The proposed mechanism for lysozyme was based on the observation by Phillips and coworkers that the only possible catalytic groups in the vicinity of the bond being cleaved were Asp-52 and Glu-35. The mechanism was actually based on the structure of the complex with tri-N-acetylglucosamine i.e. NAG3. NAG3 is a good competitive inhibitor, and was shown to bind in the A, B & C sites.

The basics of the mechanism were that

(1) binding of the sugar ring in the D-subsite resulted in distortion of the sugar conformation toward a sofa or half-chair conformation in order to relieve steric strain of the C(6)H20H group with the protein

(2) acid catalysis by Glu-35 to donate a proton to the leaving group

(3) electrostatic stabilization of the resulting oxocarbonium ion by Asp-52 (negatively charged)

(4) subsequent nucleophilic attack by water on the carbonium ion.

The mechanism may be pictured as follows:

Evidence for the mechanism

The pH dependence of lysozyme catalysis reveals that the reaction is dependent on an acid with pK ~6 and a base of pK ~ 4 in the free enzyme (from plots of kcat/Km vs. pH). The pK's for the active-site carboxyls have been measured:

Group pK of E* pK of ES Titration

Glu-35 6.0 6.6 5.9

Asp-52 3.0 3.3 4.5

* Determined from the pH-dependence of kcat/Km.

These data are from kinetic and titration measurements, and confirm the structural observations that Glu-35 is in an apolar environment (i.e. the raised pK), and Asp-52 is in a relatively polar environment. (In an apolar environment generation of charge, e.g. COO-, H+, is suppressed) Esterification of Asp-52 results in total loss of activity, as well as perturbation of the pK of Glu-35. (Water soluble carbodiimides can be used to modify all the COOH except Glu-35; this leads to inactivation. If substrate is added during the reaction Asp-52 is protected, and the resulting modified enzyme is active. Oxidation of Trp-108 leads to the formation of an oxindole which interacts with Glu-35, forming a covalent bond to the COOH and leading to inactivation.). In addition, chemical modification to the corresponding amides led to significant loss of catalytic activity, but residual unreacted enzyme may have been responsible for the observed low levels of activity.

These observations are entirely consistent with the proposed mechanism. In model reaction studies, sugar glycoside hydrolysis is acid-catalyzed; it is usual that enzyme's utilize a similar basic mechanism to the corresponding non-enzyme catalyzed reaction.

Site-directed mutagenesis in which Asp52 and Glu35 were converted to the corresponding Asn and Gln demonstrated the critical requirement for Glu35 (0% activity) and lesser essentiality of Asp52 (5% initial velocity, 0.5% SS, against cell wall substrates) (good reference: Malcolm et al., PNAS 86 133-137, 1989).

If a carbonium ion (carbocation) is involved in lysozyme catalysis then the TS structure will involve a planar, sp2-hybridized C-1. A suitable TS analog would be the lactone of NAG4

This lactone is found to be a very good competitive inhibitor with Ki = 8 x 10-8 M, compared to Km 1 x 10-5 M for the substrate NAG4. These observations are consistent with a carbonium ion TS.