Lysozyme is a natural antibacterial agent found in tears and egg whites. The hen egg white lysozyme (Mr 14,296) is a monomer with 129 amino acid residues. This was the first enzyme to have its three-dimensional structure determined, by David Phillips and colleagues in 1965. The structure revealed four stabilizing disulfide bonds and a cleft containing the active site (Fig. 6-24a; see also Fig. 4-18). More than five decades of lysozyme investigations have provided a detailed picture of the structure and activity of the enzyme, and an interesting story of how biochemical science progresses.
The substrate of lysozyme is peptidoglycan, a carbohydrate found in many bacterial cell walls (see Fig. 7-22). Lysozyme cleaves the (^1n4) glycosidic C—O bond between the two types of sugar residue in the molecule, ^-acetylmuramic acid (Mur2Ac) and ^-acetyl-glucosamine (GlcNAc), often referred to as NAM and NAG, respectively, in the research literature on enzy-mology (Fig. 6-24b). Six residues of the alternating Mur2Ac and GlcNAc in peptidoglycan bind in the active site, in binding sites labeled A through F. Model building has shown that the lactyl side chain of Mur2Ac cannot be accommodated in sites C and E, restricting Mur2Ac binding to sites B, D, and F. Only one of the bound glycosidic bonds is cleaved, that between a Mur2Ac residue in site D and a GlcNAc residue in site E. The key catalytic amino acid residues in the active site are Glu35 and Asp52 (Fig. 6-25a). The reaction is a nucleophilic substitution, with —OH from water replacing the GlcNAc at C-1 of Mur2Ac.
With the active site residues identified and a detailed structure of the enzyme available, the path to understanding the reaction mechanism seemed open in the 1960s. However, definitive evidence for a particular mechanism eluded investigators for nearly four decades. There are two chemically reasonable mechanisms that could generate the observed product of lysozyme-mediated cleavage of the glycosidic bond. Phillips and colleagues proposed a dissociative (SN1-type) mechanism (Fig. 6-25a, left), in which the GlcNAc initially dissociates in step d to leave behind a glycosyl cation (a carbocation) intermediate. In this mechanism, the departing GlcNAc is protonated by general acid catalysis by Glu35, located in a hydrophobic pocket that gives its carboxyl group an unusually high pKa. The carbocation is stabilized by resonance involving the adjacent ring oxygen, as well as by electrostatic interaction with the negative charge on the nearby Asp52. In step (2),wa-ter attacks at C-1 of Mur2Ac to yield the product. The alternative mechanism (Fig. 6-25a, right) involves two consecutive direct-displacement (SN2-type) steps. In step ®, Asp52 attacks C-1 of Mur2Ac to displace the GlcNAc. As in the first mechanism, Glu35 acts as a general acid to protonate the departing GlcNAc. In step (2), water attacks at C-1 of Mur2Ac to displace the Asp52 and generate product.
The Phillips mechanism (SN1), based on structural considerations and bolstered by a variety of binding studies with artificial substrates, was widely accepted for more than three decades. However, some controversy persisted and tests continued. The scientific method sometimes advances an issue slowly, and a truly insightful experiment can be difficult to design. Some early arguments against the Phillips mechanism were suggestive but not completely persuasive. For example, the half-life of the proposed glycosyl cation was estimated to be 10~12 seconds, just longer than a molecular vibration and not long enough for the needed diffusion of other molecules. More important, lysozyme is a member of a family of enzymes called "retaining glycosidases," all of which catalyze reactions in which the product has the same anomeric configuration as the substrate (anomeric configurations of carbohydrates are examined in Chapter 7), and all of which are known to have reactive covalent intermediates like that envisioned in the alternative (SN2) pathway. Hence, the Phillips mechanism ran counter to experimental findings for closely related enzymes.
A compelling experiment tipped the scales decidedly in favor of the SN2 pathway, as reported by Stephen Withers and colleagues in 2001. Making use of a mutant enzyme (with residue 35 changed from Glu to Gln) and artificial substrates, which combined to slow the rate of key steps in the reaction, these workers were able to stabilize the elusive covalent intermediate. This in turn allowed them to observe the intermediate directly, using both mass spectrometry and x-ray crystallography (Fig. 6-25b).
Is the lysozyme mechanism now proven? No. A key feature of the scientific method, as Albert Einstein once summarized it, is "No amount of experimentation can ever prove me right; a single experiment can prove me wrong." In the case of the lysozyme mechanism,
FIGURE 6-24 Hen egg white lysozyme and the reaction it catalyzes.
(a) Ribbon diagram of the enzyme with the active-site residues Glu35 and Asp52 shown as blue stick structures and bound substrate shown in red (PDB ID 1LZE). (b) Reaction catalyzed by hen egg white lysozyme. A segment of a peptidoglycan polymer is shown, with the lysozyme binding sites A through F shaded. The glycosidic C—O bond between sugar residues bound to sites D and E is cleaved, as indicated by the red arrow. The hydrolytic reaction is shown in the inset, with the fate of the oxygen in the H2O traced in red. Mur2Ac is N-acetylmuramic acid; GlcNAc, N-acetylglucosamine. RO— represents a lactyl (lactic acid) group; —NAc and AcN—, an N-acetyl group (see key).
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