Enzyme Mechanism - Examples
How do enzymes function at high temperature? My final enzyme mechanism example is of a comparison of a protein factor involved in protein synthesis called elongation factor (EF-Tu). 3-D structures are available for EF-Tu from E. Coli and a thermophile, Thermus aquaticus. EF-Tu binds GTP and a t-RNA/AA molecule before it binds to the ribosome. After it binds to the ribosome, GTP is hydrolyzed to GDP and the EF-Tu/GDP complex is released leaving behind the t-RNA/AA to add an amino acid to the growing polypeptide chain on the ribosome.
Figure 27. Model of the reactions in protein synthesis catalyzed by the ribosome. The role of EF-Tu is shown here as being involved in binding the t-RNA-AA complex to the ribosome, which involves the hydrolysis of GTP, during elongation of nascent polypeptide chain.
EF-Tu resembles other 'G-proteins' like those involved in cancerous cells.
Figure 28. 3-D Model showing EF-Tu domain which resembles a 'G-protein' with GDP bound to it along with the Mg2+ which is bound to the GDP.
Interestingly, the GTP hydrolysis catalyzed by EF-Tu involves a catalytic triad very similar to that of serine protease, except that it activates water to hydrolyze GTP to GDP and Pi.
Figure 29. Detail of the binding of the GDP-inhibitor bound in the active site shown the 'catalytic triad' including a water molecule which attacks the GTP molecule leading to its hydrolysis.
Figure 28, 29 & 30 are from Structure 1: 35-50 (1993).
Basically the enzyme from E coli (a mesophilic organism - grows at 37 degrees C) is exactly the same shape as the enzyme from Thermus aquaticus (a thermophile which grows at 70 degrees C). A comparison of the amino acid sequence shows they are 70% the same. However, the thermophilic enzyme 3 insertions of amino acids in the sequence. One of these insertions is 10 AA's long. It seems that these extra AA's are all that is needed to make the thermophilic enzyme stable at high temperature.
Figure 30. Amino acid sequence of 2 thermophilic forms of EF-Tu compared to the sequence of EF-Tu from E. coli. The amino acids which are the same in all three sequences are shown on the 4th line of each segment of the sequence. The extra 10 amino acids which is the major difference between the enzyme forms stable at the higher temperatures of the thermophiles is lined above and below.
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu