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Catalysis by Hen Egg-White Lysozyme Proceeds Via a Covalent Intermediate

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Catalysis by Hen Egg-White Lysozyme Proceeds Via a Covalent Intermediate This is a repository copy of Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/131/ Article: Vocadlo, D J, Davies, G J orcid.org/0000-0002-7343-776X, Laine, R et al. (1 more author) (2001) Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate. Nature. pp. 835-838. ISSN 0028-0836 https://doi.org/10.1038/35090602 Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ letters to nature ................................................................. measuring a-deuterium kinetic isotope effects (kH/kD, where kH and kD are rate constants for reactions of protium- and deuterium- Catalysis by hen egg-white lysozyme labelled substrates) with substrates for which the breakdown of the proceeds via a covalent intermediate intermediate is rate determining. In all of these cases, kH/kD has been found to be greater than unity6,8. Effects greater than unity, David J. Vocadlo , Gideon J. Davies ², Roger Laine³ measured on the second step, indicate that the anomeric centre * * 3 2 & Stephen G. Withers* undergoes rehybridization from sp in the intermediate to sp in the transition state. These results are consistent only with the collapse of * Protein Engineering Network of Centres of Excellence and a tetrahedral covalent intermediate through an oxocarbenium ion- the Department of Chemistry, University of British Columbia, Vancouver, like transition state. A long-lived ion pair may not be reconciled British Columbia V6T 1Z1, Canada with these observations. ² Department of Chemistry, Structural Biology Laboratory, University of York, In order to observe the covalent intermediate experimentally, a Heslington, York YO10 5DD, UK situation in which the rate of formation (k2) of the intermediate is ³ Departments of Biology and Chemistry, Louisiana State University, signi®cantly greater than its rate of breakdown (k3) must be created Baton Rouge, Louisiana 70808, USA (Fig. 1). Considerable efforts have previously been made, with .............................................................................................................................................. HEWL, to ®nd such a condition yet in no case have these efforts Hen egg-white lysozyme (HEWL) was the ®rst enzyme to have its proven successful9,10. This observation strongly suggests that, for three-dimensional structure determined by X-ray diffraction HEWL, hydrolysis of the intermediate (k3) occurs at a much greater 1 techniques . A catalytic mechanism, featuring a long-lived oxo- rate than does its formation (k2). We used a mutant enzyme in carbenium-ion intermediate, was proposed on the basis of model- which the catalytic acid/base carboxylate residue had been replaced building studies2. The `Phillips' mechanism is widely held as the by the corresponding amide: the HEWL(E35Q) mutant enzyme. paradigm for the catalytic mechanism of b-glycosidases that Mutation of the catalytic acid/base residue of HEWL and other b- cleave glycosidic linkages with net retention of con®guration of retaining glycosidases has been shown to greatly reduce the rate of the anomeric centre. Studies with other retaining b-glycosidases, hydrolysis of unactivated glycosides by slowing both steps of the however, provide strong evidence pointing to a common mechan- reaction11,12. Substrates bearing activated leaving groups do not ism for these enzymes that involves a covalent glycosyl-enzyme require signi®cant acid catalytic assistance and so react at near wild- intermediate, as previously postulated3. Here we show, in three type rates to yield the glycosyl-enzyme intermediate12. Hydrolysis different cases using electrospray ionization mass spectrometry, a of this intermediate, however, is slowed enormously as the attack of catalytically competent covalent glycosyl-enzyme intermediate water no longer bene®ts from the base catalysis ordinarily pro- during the catalytic cycle of HEWL. We also show the three- vided by the enzyme. Indeed, on incubation of HEWL(E35Q) with dimensional structure of this intermediate as determined by X- ray diffraction. We formulate a general catalytic mechanism for all retaining b-glycosidases that includes substrate distortion, O formation of a covalent intermediate, and the electrophilic Glu 35 migration of C1 along the reaction coordinate. O OH The natural substrate of HEWL is the peptidoglycan cell wall of H O O O NAM-OR Gram-positive bacteria. It is composed of crosslinked oligosacchar- HO HO NHAc RO-NAG ides consisting of alternating 2-acetamido-2-deoxy-glucopyrano- O O side (NAG) and 2-acetamido-2-deoxy-3-O-lactyl-glucopyranoside 'RO (NAM) residues. In the textbook mechanism proposed by Phillips AcHN O O (Fig. 1, path B), the enzyme cleaves the substrate by ®rst binding the Asp 52 polysaccharide in a cleft six saccharide units long (A±F or -4 to +2). Path A Path B k k The substrate was proposed to bind in a distorted conformation in 2 2 which a NAM unit in the -1 (D) subsite adopts a half-chair O O conformation, thereby forming a kink in the oligosaccharide Glu 35 Glu 35 between sites -1 and +1 (D and E). A catalytic acid/base, Glu 35, O O H was suggested to protonate the glycosidic oxygen while Asp 52 H NAG-OR NAG-OR O stabilizes the developing oxocarbenium ion intermediate through O HO HO RO-NAG electrostatic interactions. It was also proposed that Asp 52 acts to RO-NAG O O O O 'RO shield the a-face of the glycosyl oxocarbenium ion, such that it can 'RO AcHN O O only be intercepted by water from the b-face to yield the hydrolysed AcHN O O product with retained stereochemistry at C1. It was later demon- Asp 52 strated that the intermediate could also be intercepted by another Asp 52 saccharide moiety that had diffused into the active site, producing a k k H2O transglycosylation product4. H2O 3 3 O The nature and purported stability of the ion-pair intermediate Glu 35 NAG-OR have always been points of contention. The lifetime of the glycosyl NAG-OR O - cation in water (10 12 s) is estimated to be just slightly longer than a H bond vibrationÐmuch less than the time required for diffusion O H 5 HO events . Furthermore, there is no experimental evidence for a long- RO-NAG O O lived ion-pair intermediate at the active site of any glycosidase, 'RO including HEWL. Conversely, for every retaining b-glycosidase AcHN O O studied in detail to date6, the intermediate has been shown to be a reactive, covalent acylal ester or, in the case of retaining Asp 52 b-hexosaminidases using neighbouring-group participation, a covalent oxazoline intermediate7. The most rigorous demonstration Figure 1 Two possible catalytic mechanisms for HEWL. Path A; the Koshland mechanism; of the covalent nature of the intermediate has been obtained by path B; the Phillips mechanism. R, oligosaccharide chain; R9, peptidyl side chain. NATURE | VOL 412 | 23 AUGUST 2001 | www.nature.com © 2001 Macmillan Magazines Ltd 835 letters to nature 13 -1 -1 -1 -1 chitobiosyl ¯uoride (NAG2F) we observed, using electrospray 6 0.1 M min and kcat/Km = 1,200 M min , respectively, where ionization mass spectrometry (ESI-MS), a mixture of the free kcat is the rate constant when both substrates are at saturating enzyme HEWL(E35Q) and a high steady-state population concentrations and Km is the Michaelis constant) but still turned -1 (Fig. 2b) of a covalent intermediate (relative molecular mass (Mr) over too rapidly (k3 . 96 min ) for X-ray crystallographic studies. 14,719) with a mass increase (405) revealing the attachment of a By combining these two completely independent approaches, we chitobiosyl moiety (Mr 407) to HEWL. This intermediate turned characterized the covalent glycosyl-enzyme intermediate formed on over too rapidly for X-ray analysis, so a complementary strategy was lysozyme by X-ray crystallography. On incubation of HEWL(E35Q) sought. with NAG2FGlcF, the stoichiometric accumulation of a covalent To reduce the rate of the second step in a different approach, we intermediate could be observed by ESI-MS (Fig. 2d). The second- incorporated ¯uorine in place of the 2-acetamido group of order rate constant governing the formation of the intermediate -1 -1 chitobiose, while keeping a good leaving group at the anomeric (ki/Ki = 0.20 6 0.05 M min ) is only 50-fold less than that centre. Previous studies, designed to rule out a possible mechanism observed for the wild-type enzyme interacting with the same involving anchimeric assistance from the 2-acetamido group, substrate. Hydrolysis of the covalent intermediate, however, was 5 -4 -1 demonstrated that substrates bearing hydrogen and hydroxyl sub- slowed by at least 5.3 ´ 10 -fold (k3 , (1.8 6 0.6) ´ 10 min ) with stitutions at this centre are hydrolysed at similar rates to that of the the result that the covalent glycosyl-enzyme intermediate was stable parent compound bearing the acetamido group, and thus reveal that for several days (half-life t1/2 =64h)at258C. The HEWL(E35Q) the amide functionality is not critical for catalysis14,15. An electro- regenerated on such turnover could once again be labelled by negative ¯uorine substituent at the 2 position will, however, incubating it with NAG2FGlcF (data not shown), indicating that inductively destabilize the oxocarbenium ion-like transition states the formation and breakdown of the intermediate leaves the enzyme leading to the formation and hydrolysis of the intermediate, slowing unchanged and catalytically competent.
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