Lysozyme (covalent intermediate) template

Hen egg-white lysozyme cuts the β(1→4) bond between NAM (site D) and NAG (site E) of bacterial peptidoglycan: Glu35 (acid) protonates the leaving oxygen while Asp52 attacks C1 to form an α-glycosyl-enzyme ester; Glu35 (now a base) activates water, which displaces Asp52 to give the β-hemiacetal.

Mechanism step by step

Glu35 protonates the glycosidic oxygen as Asp52 displaces it; water then hydrolyses the glycosyl-enzyme with overall retention

1. Glycosyl-enzyme – Glu35–H protonates the glycosidic O as Asp52 attacks C1 from the opposite face (inversion: α-ester)
2. Hydrolysis – Glu35⁻ activates water, which attacks C1 and displaces Asp52 (second inversion: β)

Key points

• Two inversions give overall retention at C1, the hallmark of retaining β-glycosidases (Koshland double displacement).
• Phillips (1966) proposed a long-lived oxocarbenium-ion intermediate stabilised by Asp52; Vocadlo et al. (2001) trapped the covalent α-glycosyl-Asp52 ester by mass spectrometry and X-ray (E35Q mutant, 2-fluoro sugars).
• Both transition states still have oxocarbenium-ion character, and binding in site D distorts the NAM ring towards a half-chair.
• Glu35 sits in a non-polar region (pKa ≈ 6), so it stays protonated; Asp52 is in a polar region and ionised.

Drawn with 142 atoms, 9 arrows, 6 shapes – every one editable in MolDraw.

Sources

• Nelson & Cox, Lehninger Principles of Biochemistry, 8th ed., 2021, ch. 6 Textbook
• Voet & Voet, Biochemistry, 4th ed., Wiley 2011, ch. 15 Textbook
• Phillips, Sci. Am. 1966, 215 (5), 78 Original paper
• Vocadlo, Davies, Laine & Withers, Nature 2001, 412, 835 Original paper