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Download Jmol-14.29.29-binary.zip

Uncompress the zip file

Double-click on Jmol.jar in the uncompressed directory

 

Download Molecular_modeling2_pdb_files.zip and uncompress

 

File --> Console to get command line.

Commands to be typed in the console window will be listed in Courier.

 

Open files by going to File menu, then Open (File --> Open)

                         

Right-Click (PC) or Control-click (Mac) to get pop-up menu.

Go to Style in the pop-up.

Menu commands will be listed in Helvetica

Style --> Scheme --> Ball and Stick

 

In the image window, figure out how to zoom in and out, and how to rotate the molecules. You should zoom and rotate ALL the molecules you examine after each display change.

 

2-fluorolactose

 

Go to File menu, then Open (File --> Open)

Select fluorolactose.pdb from the uncompressed Molecular_modeling2_pdb_files directory

 

Style --> Scheme --> Ball and Stick

Style --> Scheme --> CPK Spacefill

 

Examine the 2-fluorolactose structure. This molecule has the 2' OH replaced with a highly electronegative fluorine atom, making hydrolysis of the C1-O1 bond unfavorable.

 

1. In the unbound state, the polar atoms interact with polar water molecules. The protein must minimally compensate for these lost interactions in order to bind. What additional interactions must be made by the protein to drive binding?

 

b-galactosidase holo complex

 

File --> Open fluorolactose_holo_complex.pdb

 

Style --> Scheme --> Cartoon

color chain

Style --> Scheme --> CPK Spacefill

color chain

 

Note the extensive interfaces between the different subunits of the tetramer.

 

select LAC

Style --> Scheme --> CPK Spacefill

 

Note that each lactose is bound primarily by a single subunit.

 

1. b-galactosidase is described well by the hyperbolic curve of Michaelis-Menten. What does this suggest about interactions between the four active sites in the tetramer?  Is this more like oxygen binding to myoglobin or oxygen binding to hemoglobin?

 

b-galactosidase active site

 

File --> Open fluorolactose_complex.pdb

 

Style --> Scheme --> Cartoon

color chain

 

select LAC

Style --> Scheme --> CPK Spacefill

restrict LAC

select within (5.0, LAC ) and sidechain and not LAC

Style->Scheme->Ball and stick

View --> Define Center

color cpk

select MG

Style->Scheme->CPK Spacefill

color purple

 

1. What sidechains (amino acid and number) might be involved in driving binding of lactose?

 

2. From the pH dependence of b-galactosidase activity, most students found pK1 ~ 5.5 and pK2 ~ 8.5. From the structure, what sidechains (amino acid and number) might need to be deprotonated for catalysis?

 

3. Inhibitor II from Session 7 is IPTG, another non-hydrolysable lactose analog. Most students found this to be a competitive inhibitor. Why might this be the case?

 

b-galactosidase covalent intermediate

 

File --> Open covalent_intermediate.pdb

 

This structure contains fluorinated galactose, essentially half of the fluorolactose.  

 

Style --> Scheme --> Cartoon

color chain

 

select GAL

Style --> Scheme --> CPK Spacefill

Style->Scheme->Ball and stick

 

restrict GAL

select within (5.0, GAL ) and sidechain and not GAL

Style->Scheme->Ball and stick

View --> Define Center

color cpk

select GAL

Style --> Scheme --> CPK Spacefill

 

1. What happened to oxygen OE2 of Glu 537?

 

select MG

Style->Scheme->CPK Spacefill

color purple

select HOH

Style->Scheme->CPK Spacefill

color cyan

 

2. The fluorinated galactose was delivered to the enzyme with a very strong leaving group (dinitrophenol) that permits the formation of this covalent intermediate. However, the strong electronegativity of the fluorine prevents moving past this step. Inhibitors such as this are often called "suicide inhibitors." What mode of inhibition do you expect, competitive or non-competitive? Why?

 

3. Mg2+ is often surrounded by a hydration shell of several water molecules (HOH). What might be the role of the displayed cyan water molecule? Which carbon atom might it react with?

 

4. Inhibitor I from Session 7 is EDTA, an organic compound that sequesters divalent cations such as Mg2+. Most students found this to be an un-competitive or mixed inhibitor. Why might this be the case?

 

 

 

 

 

 

 

Navin Pokala, PhD
navin.pokala at nyit dot edu

Department of Biological and Chemical Sciences
New York Institute of Technology
Theobald Science Center, Room 423
​Old Westbury, NY 11568-8000
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