17 Aug Question: This Problem Is About The Relationship Between The Structure And Function Of The Enzyme Phytase, Which Catalyzes The Hydrolysis Of Phosphoester Linkages In Phytate, Otherwise Known As Myo-inositol-1,2,3,4,5,6-hexaphosphate: Phytate + 6 H2O ® Inositol + 6 Pi (inorganic Phosphate) The Molecule Myo-inositol (hereafter Simply “inositol”) Is A Part …
This problem is about the relationship between the structure and function of the enzyme phytase, which catalyzes the hydrolysis of phosphoester linkages in phytate, otherwise known as myo-inositol-1,2,3,4,5,6-hexaphosphate:
phytate + 6 H2O ® inositol + 6 Pi (inorganic phosphate)
The molecule myo-inositol (hereafter simply “inositol”) is a part of several important biomolecules, primarily phospholipids (see Chapter 11). Plants use phytate to store intracellular phosphate. One of the problems in agriculture is that non-ruminant animals (e.g., pigs and chickens) cannot digest phytate, necessitating phosphate supplementation. It also creates a downstream problem, as the waste from these animals can lead to blooms of freshwater algae (which can degrade phytate). One of the solutions to this issue is a biotechnological approach, in which animal feed is supplemented with phytase enzyme, which can break down the phytate in the stomach (it has a low pH optimum). Here you will examine the structure of the bacterial enzyme.
You will have access to the crystal structures in the following PDB files:
|
PDB ID# |
Description |
|
1BPH |
Enzyme without substrate at pH 5.0 |
|
2BPH |
Enzyme bound to tungstate at pH 6.6 |
|
3BPH |
Mutant enzyme (H17A) bound to phytate at pH 5.0 |
Note that the structure of this protein was solved using isomorphous replacement with crystals bound to mercury. The Hg(II) ions are at the end of the list, typically as “HG500”, “HG501”, etc. For the purpose of this problem, please ignore the mercury ions in the structure. They are largely irrelevant to the function of the protein (which typically does not exist in conditions of high mercury). The tungstate (WO4504) in 2BPH mimics the phosphate moiety that is attacked during the reaction.
- Briefly describe the overall tertiary and quaternary structure of phytase.+3
(Use the 1BPH file for this. Approach this as you did in Problem 1.)
How many and what kind of domains does it have (e.g. what class)?
- Compare the structure of phytase in the absence and presence of bound phytase, by comparing the structures in 1BPH and 3BPH. Note that 3BPH made use of a mutant in which the active site histidine, which serves as the primary nucleophile in the enzyme, was converted to alanine; “H17A” means that His17 was converted to Ala. (Otherwise the enzyme would bind phytate and then hydrolyze it.)
Are the any major conformational changes that occur upon binding phytate?
If so, describe them. +2
Briefly describe where the phytate binding site is located, relative to the domain(s) of the protein. +2
Identify at least 6 residues that are important for binding phytate. +1.5 each
|
Residue |
Atom comes from which part of the residue? Interacts with which atom of phytate? |
distance between atoms (Å) |
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