Chemistry Department University of Southern Maine
Links To Files Used In Biochemistry Class (CHY 361-363)
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Topic: Monosaccharide Metabolism
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Molecules To Explore
FBP, Mg2+, and ADP in PFK-1 (Convergent Stereo)
Phosphofructokinase, Complex With Products and Allosteric Effector ADP
Phosphofructokinase (PFK-1) is the pacemaker enzyme of glycolysis. Here is a model of two subunits of the tetrameric enzyme from E. coli: 1PFK.pdb.
At the active sites are the products, fructose-1,6-bisphosphate (FBP) and Mg2+/ADP. The crystallographers attempted to crystallize the enzyme with fructose-1-phosphate and ADP, which should produce an inactive complex. To their surprise, the electron-density map clearly showed FBP instead. They suggest that it formed by reaction with contaminating ATP.
To see the full tetramer of PFK-1, click here. This is a large file (~800K), and may take several minutes to download.
Think About It
· Two molecules of ADP are bound to each subunit. One ADP is bound adjacent to FBP at the active site. The second ADP is far away from this site. What is the role of the second ADP- binding site?
· Examine the binding of Mg2+ to all four ADP's in the dimer. Does the ion play the same role in all cases?
Glyceraldehyde-3-Phosphate Dehydrogenase, NAD Binding Site
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)catalyzes the reversible oxidation of G3P by NAD+. Here is a model of the dimeric GAPDH from E. coli: 1GAD.pdb.
Explore the NAD binding site. Find the catalytic side chain of CYS149 near the nicotinamide ring.
Think About It
· Display the backbone of the protein and color by secondary structure. What type of structure forms the interface between the subunits?
· Using your text, learn how to distinguish the A and B faces of the nicotinamide ring (in LNC, see page 391). Determine whether GAPDH is an A-side or B-side NAD dehydrogenase. Confirm your answer in your text (LNC, page 392).
· The N313T site-directed mutant form of GAPDH exhibits weaker binding of NAD and lower catalytic efficiency. Look at the position of ASN313 with respect to NAD. What kind of interaction occurs between them? What role might ASN313 play in binding and catalysis? If you use SwissPdbViewer, you can compare the mutant with this model. Click here to obtain the coordinate file for the N313T mutant from the Protein Data Bank.
Pyruvate Decarboxylase (Yeast), TPP Binding Site
Pyruvate decarboxylase contains the cofactor thiamine pyrophosphate (TPP). The active form of TPP is the conjugate base, resulting from loss of a proton from C-2, the carbon between N and S in the thiazole ring. Here is a model of the dimeric enzyme from yeast: 1PVD.pdb (~800K).
Like many di- and tri-phosphates, TPP is bound to its enzyme as a complex with Mg2+. Restrict your view to atoms within 7 or 8 angstroms of TPP in chain a [restrict within (7.0, tpp:a)] to see the binding site for TPP. Display the alpha carbons of chains a and b in differenct colors and notice that the TPP binding site includes residues from both subunits. The active sites in this dimer lie at the subunit interfaces.
Think About It
· Can you find a nearby group that might be responsible for depronating C-2 of TPP? What interactions position this group for its role?
· What might be the role of the pyrophosphate/Mg2+ moiety of TPP? Hint: See LNC, pages 207 and 352.
· Would you expect a plot of rate versus substrate concentration for this enzyme to exhibit square hyperbola or sigmoid shape?
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