An allolactose trapped at the lacZ β-galactosidase active site with its galactosyl moiety in a (4)H3 conformation provides insights into the formation, conformation, and stabilization of the transition state.
When lactose was incubated with G794A-β-galactosidase (a variant with a "closed" active site loop that binds transition state analogs well) an allolactose was trapped with its Gal moiety in a (4)H3 conformation, similar to the oxocarbenium ion-like conformation expected of the transition state. The numerous interactions formed between the (4)H3 structure and β-galactosidase indicate that this structure is representative of the transition state. This conformation is also very similar to that of d-galactono-1,5-lactone, a good transition state analog. Evidence indicates that substrates take up the (4)H3 conformation during migration from the shallow to the deep mode. Steric forces utilizing His418 and other residues are important for positioning the O1 leaving group into a quasi-axial position. An electrostatic interaction between the O5 of the distorted Gal and Tyr503 as well as C-H-π bonds with Trp568 are also significant. Computational studies of the energy of sugar ring distortion show that the β-galactosidase reaction itinerary is driven by energetic considerations in utilization of a (4)H3 transition state with a novel (4)C1-(4)H3-(4)C1 conformation itinerary. To our knowledge, this is the first X-ray crystallographic structural demonstration that the transition state of a natural substrate of a glycosidase has a (4)H3 conformation.
Biochemistry Division, Faculty of Science, University of Calgary, Calgary, AB T2N 1N4, Canada.|Biochemistry Division, Faculty of Science, University of Calgary, Calgary, AB T2N 1N4, Canada.
Robert W
RW
Wheatley
Biochemistry Division, Faculty of Science, University of Calgary, Calgary, AB T2N 1N4, Canada.|Biochemistry Division, Faculty of Science, University of Calgary, Calgary, AB T2N 1N4, Canada.
Reuben E
RE
Huber
D019943:Amino Acid Substitution; D001665:Binding Sites; D055162:Biocatalysis; D002236:Carbohydrate Conformation; D020134:Catalytic Domain; D019295:Computational Biology; D030562:Databases, Protein; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D007763:Lac Operon; D007785:Lactose / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D055672:Static Electricity; D013237:Stereoisomerism; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D006639:Histidine; C001970:allolactose; D001616:beta-Galactosidase; D007785:Lactose
conformation; mechanism; mécanisme; transition state; tryptophan; tryptophane; tyrosine; était de transition; β-galactosidase