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Directed "in situ" inhibitor elongation as a strategy to structurally characterize the covalent glycosyl-enzyme intermediate of human pancreatic alpha-amylase.

Zhang R, Li C, Williams LK, Rempel BP, Brayer GD, Withers SG
Biochemistry 48 10752-64 (2009)
Related entries: 3ij8, 3ij9

Cited: 15 times
EuropePMC logo PMID: 19803533

Abstract

While covalent catalytic intermediates of retaining alpha-transglycosylases have been structurally characterized previously, no such information for a hydrolytic alpha-amylase has been obtained. This study presents a new "in situ" enzymatic elongation methodology that, for the first time, has allowed the isolation and structural characterization of a catalytically competent covalent glycosyl-enzyme intermediate with human pancreatic alpha-amylase. This has been achieved by the use of a 5-fluoro-beta-l-idosyl fluoride "warhead" in conjunction with either alpha-maltotriosyl fluoride or 4'-O-methyl-alpha-maltosyl fluoride as elongation agents. This generates an oligosaccharyl-5-fluoroglycosyl fluoride that then reacts with the free enzyme. The resultant covalent intermediates are extremely stable, with hydrolytic half-lives on the order of 240 h for the trisaccharide complex. In the presence of maltose, however, they undergo turnover via transglycosylation according to a half-life of less than 1 h. Structural studies of intermediate complexes unambiguously show the covalent attachment of a 5-fluoro-alpha-l-idosyl moiety in the chair conformation to the side chain of the catalytic nucleophile D197. The elongated portions of the intermediate complexes are found to bind in the high-affinity -2 and -3 binding subsites, forming extensive hydrogen-bonding interactions. Comparative structural analyses with the related noncovalent complex formed by acarbose highlight the structural rigidity of the enzyme surface during catalysis and the key role that substrate conformational flexibility must play in this process. Taken together, the structural data provide atomic details of several key catalytic steps. The scope of this elongation approach to probe the active sites and catalytic mechanisms of alpha-amylases is further demonstrated through preliminary experiments with porcine pancreatic alpha-amylase.

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  2. Crystal structure of the Chlamydomonas starch debranching enzyme isoamylase ISA1 reveals insights into the mechanism of branch trimming and complex assembly. Sim L, Beeren SR, Findinier J, Dauvillée D, Ball SG, Henriksen A, Palcic MM. J Biol Chem 289 22991-23003 (2014)
  3. Structural insight into how Streptomyces coelicolor maltosyl transferase GlgE binds α-maltose 1-phosphate and forms a maltosyl-enzyme intermediate. Syson K, Stevenson CE, Rashid AM, Saalbach G, Tang M, Tuukkanen A, Svergun DI, Withers SG, Lawson DM, Bornemann S. Biochemistry 53 2494-2504 (2014)
  4. Computer simulations explain the anomalous temperature optimum in a cold-adapted enzyme. Sočan J, Purg M, Åqvist J. Nat Commun 11 2644 (2020)
  5. Mechanistic analysis of trehalose synthase from Mycobacterium smegmatis. Zhang R, Pan YT, He S, Lam M, Brayer GD, Elbein AD, Withers SG. J Biol Chem 286 35601-35609 (2011)
  6. Activity-Based Protein Profiling of Retaining α-Amylases in Complex Biological Samples. Chen Y, Armstrong Z, Artola M, Florea BI, Kuo CL, de Boer C, Rasmussen MS, Abou Hachem M, van der Marel GA, Codée JDC, Aerts JMFG, Davies GJ, Overkleeft HS. J Am Chem Soc 143 2423-2432 (2021)
  7. Aromatic interactions at the catalytic subsite of sucrose phosphorylase: their roles in enzymatic glucosyl transfer probed with Phe52→Ala and Phe52→Asn mutants. Wildberger P, Luley-Goedl C, Nidetzky B. FEBS Lett 585 499-504 (2011)
  8. Letter Glucosyl epi-cyclophellitol allows mechanism-based inactivation and structural analysis of human pancreatic α-amylase. Caner S, Zhang X, Jiang J, Chen HM, Nguyen NT, Overkleeft H, Brayer GD, Withers SG. FEBS Lett 590 1143-1151 (2016)
  9. Structural, mechanistic, and computational analysis of the effects of anomeric fluorines on anomeric fluoride departure in 5-fluoroxylosyl fluorides. Lee SS, Greig IR, Vocadlo DJ, McCarter JD, Patrick BO, Withers SG. J Am Chem Soc 133 15826-15829 (2011)
  10. Structural insights into the catalytic reaction that is involved in the reorientation of Trp238 at the substrate-binding site in GH13 dextran glucosidase. Kobayashi M, Saburi W, Nakatsuka D, Hondoh H, Kato K, Okuyama M, Mori H, Kimura A, Yao M. FEBS Lett 589 484-489 (2015)
  11. Structure-function analysis of silkworm sucrose hydrolase uncovers the mechanism of substrate specificity in GH13 subfamily 17 exo-α-glucosidases. Miyazaki T, Park EY. J Biol Chem 295 8784-8797 (2020)
  12. Conformational Itinerary of Sucrose During Hydrolysis by Retaining Amylosucrase. Alonso-Gil S, Coines J, André I, Rovira C. Front Chem 7 269 (2019)
  13. α-Amylase Modulation: Discovery of Inhibitors Using a Multi-Pharmacophore Approach for Virtual Screening. Al-Asri J, Gyémánt G, Fazekas E, Lehoczki G, Melzig MF, Wolber G, Mortier J. ChemMedChem 11 2372-2377 (2016)
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  15. Lavandula angustifolia mill. (Lamiaceae) ethanol extract and its main constituents as promising agents for the treatment of metabolic disorders: chemical profile, in vitro biological studies, and molecular docking. Tundis R, Grande F, Occhiuzzi MA, Sicari V, Loizzo MR, Cappello AR. J Enzyme Inhib Med Chem 38 2269481 (2023)


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