1 Chem451/553 Problem Set #1 1. Convert the following glycan structures to the indicated format: A) Draw the chemical structure of the following glycan:...

honework problem set


1 Chem451/553 Problem Set #1 1. Convert the following glycan structures to the indicated format: A) Draw the chemical structure of the following glycan: Neu5Aca2-3Galb1-4(Fuca1-3)GlcNAcb1-4Galb1-3GlcNAc B) The coloured cartoon (shapes) of the following N-glycan crystalized on a protein (shown in electron density) with linkages (note: Asp402 is the site of glycosylation). C) Convert this cartoon representation of an O-glycan to its written form and describe what ‘core’ it is: 2 2. From the following N-glycan, answer the following question: a) How many inverting glycosyltransferases were involved in making it? b) How many retaining glycosyltransferases were involved in making it? c) From the point of glucose entering the cell, how many moles of ATP are required to make 1 mole of this glycan? d) Describe 3 ways in which the biosynthesis of this N-glycan on proteins helped the protein to which it was attached fold correctly. 3 3. A protein has 4 N-glycosylation sites at Asn 31, 129, 256, and 321. Researchers have carefully been able to quantify the N-glycans at each site from protein purified from mammalian cells: Asn31 Asn129 Asn256 Asn321 100% site occupancy 87% site occupancy 100% occupancy 75% occupancy 25% high mannose 22% high mannose 93% high mannose 21% high mannose 2% hybrid 3% hybrid 2% hybrid 56% hybrid 73% complex type 75% complex type 5% complex type 23% complex type Furthermore, the researchers performed site-directed mutagenesis to create four different proteins with individual AsnàAla mutations and observed the following protein expression levels of the mutant protein relative to the WT protein: N29A N131A N283A N494A 7% 97% 12% 96% Answer the following questions: A) What do the expression levels of the mutants and site occupancy strongly suggest as to the importance of N-glycans at each position? B) Propose a molecular mechanism to account for the different patterns of glycosylation. Be specific. C) If cells were treated with kifunensin, how would site occupancy and the type of N-glycan change at each position? D) Researchers wish to express and purify the protein from bacteria instead. However, they are disappointed that their yield of this protein is nearly zero. Why? (hint: bacteria do not have an ER/Golgi) 4 4. Core fucosylation is catalyzed by a fucosyltransferase 8 (Fut8) in humans. The product is a1-6 linked fucose to the first N-acetylglucosamine residue on N-glycans. a) Draw out the product of the reaction: b) Mutational analysis of Fut8 found that Asp272 is critical for catalysis. Proposal a catalytic mechanism for Fut8 using GFP-Fuc and GlcNAc as the substrates and show the role played by Asp272. c) Propose a transition state for the enzyme-catalyzed reaction. Include partial charges. d) Based on your transition state and taking only into account electronic considerations, would you expect GDP-2-fluoro-fucose to be a better or worse substrate than GDP-Fuc? Explain 5 5. Consider one of the enzymes involved in biosynthesis of GDP-Fucose: GDP 6-deoxy-4-keto-mannose 3,5- epimerase, which proceeds through 4 steps and 3 discrete intermediates. The first 2 steps involve two key residues in the enzyme active site that play the role of an acid/base, wherein the first intermediate is an enolate: a) Draw the first intermediate in the space above as well as arrows to depict the flow of electrons b) Propose a name for the 2nd intermediate based on the 6-carbon aldoses introduced in the first lecture. c) The 3rd and 4th steps proceed through a similar series of steps to get to the final product. Draw these last two steps, including the 2nd and 3rd intermediate. d) Cells can also salvage free fucose from the environment. There are 2 steps in activating Fucose to GDP- Fucose. Draw out these chemical steps. e) Bonus: Propose a fun name for the first enzyme involved in the 1st step of the salvage pathway. 6 6. Draw the acid-catalyzed anomerization of xylose from its b to a anomer showing any intermediates, products, and be sure to use arrow pushing. 7. Excess glucose in blood causes pathologies in Diabetes. Glucose can react with a protein and then go on to form many re-arranged products, the simplest of which is oxidation by molecular oxygen to form carboxylmethyl-lysine (HOOC-CH2-lysine) on the target protein and erythronic acid. Propose a reaction mechanism in going from glucose and a protein to the described products.