Mechanism of glycoside hydrolase family 31 : mechanistic plasticity of glycosidic bond cleavage
| Autor: | Lee, Seung Seo |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2004 |
| Druh dokumentu: | Text |
| Popis: | Glycoside hydrolase. (GH) family 31 contains enzymes that catalyze several different reactions on glycosides. These include a nucleophilic substitution reactions with net retention of stereochemistry, common to retaining glycosidases, as well as an unusual β-elimination reaction. Since structures and mechanism are expected to be conserved in the same gene family, the two mechanisms were expected to feature common aspects. Three different GH family 31 enzymes were therefore studied. A double displacement mechanism for the retaining α-glucosidase from Aspergillus niger was shown via trapping of a covalent glycosyl-enzyme intermediate with the mechanism based inactivator 5-fluoro-α-D-glucopyranosyl fluoride. The amino acid residue involved, Asp 224, was identified by LC MS/MS analysis of proteolytic digests. This residue is fully conserved in GH family 31 and has been suggested to be the catalytic nucleophile. An unusual GH family 31 enzyme, the α-1,4-glucan lyase from Gracilariopsis sp. (GLase) that cleaves the glycosidic bond of α-1,4-glucans via a net β-elimination reaction was also studied. The trapping of a covalent glycosyl-enzyme intermediate using 5-fluoro-(β-L-idopyranosyl fluoride, another mechanism based inactivator of α-glucosidases, strongly suggests that the mechanism also involves the formation of a covalent intermediate like that of α-glucosidases. The labeled amino acid residue was confirmed to be the highly conserved Asp 553, equivalent to Asp 224, the catalytic nucleophile in α-glucosidase from A. niger. A detailed mechanistic evaluation was also carried out. A classical bell shaped pH dependence of k[sub cat]/K[sub m] indicates two ionizable groups (pK[sub α1] =3.1, pK[sub α2] = 6.7). Brønsted relationships of log k[sub cat] versus K[sub m] and log (k[sub cat]/K[sub m]) versus pK[sub a] for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK[sub a] with low β[sub 1g] values of -0.32 and -0.33, respectively. The combination of these low β[sub 1g] values with large a-secondary deuterium kinetic isotope effects (k[sub H]/k[sub D] = 1.16 ~ 1.19) on the first step indicate a transition state for the glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen. Substantial oxocarbenium ion character at the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-α-D-glucopyranosyl fluoride, was the second, elimination, step shown to be rate-limiting. The large a-secondary deuterium kinetic isotope effect (k[sub H]/k[sub D] = 1.23) at C1 and the small primary deuterium kinetic isotope effect (k[sub H]/k[sub D] = 1.92) at C2 confirm an E2 mechanism with considerable E1 character for this second step. This considerable structural and mechanistic similarity with retaining a-glucosidases is a clear example of the mechanistic plasticity of glycosidic bond cleavage through evolution. Finally, an unknown protein (yic1) whose sequence has high similarity with GH family 31 was cloned from E. coli. and shown to be an α-xylosidase. Two new mechanism-based inactivators for α-xylosidases, (5S)- and (5R)-5-fluoro-α-Dxylopyranosyl fluorides were synthesized and shown to inactivate this enzyme. The amino acid residue labeled by these inactivators was identified as the invariant catalytic aspartate residue Asp 416, demonstrating the integrity of the mechanism within this gene family. Science, Faculty of Chemistry, Department of Graduate |
| Databáze: | Networked Digital Library of Theses & Dissertations |
| Externí odkaz: |
|