Enzyme Mechanism - Examples
Part II. pH Effects in Active Sites
Why does the activity of enzymes vary with pH?
Changing the pH alters the ionization state of amino acid side chains that ionize:
Lys, Arg, His, Asp and Glu
So, if ionic bonds are important to structural stability then the shape of the enzyme will change and the functionality of the enzyme will change. This is a general phenomena - related to the overall 3-D structure of all enzymes.
Figure 1. pH optimum of an enzyme shown as the classic 'bell-shaped' curve.
Why is chymotrypsin most active at pH 8? What if AA side chains that ionize are involved in catalysis?
For example, the mechanism of serine proteases, which was described in some detail in lecture 15B, involves Asp-102 and His-57. A ball-and-stick model of the active site of chymotrypsin is shown below:
Figure 2. Catalytic residues in the active site of chymotrypsin with the 'catalytic triad' of Asp-102, His-57 and Ser-195 highlighted.
Figure from Voet's Biochemistry, copyright ©1990, John Wiley & Sons, Inc.
Protonation of Asp-102 and His-57 would block enzyme activity at pH below the pK of Asp (~4). But the pK of His-57 in chymotrypsin is ~6.8, a bit higher than a normal His (~6). So, we expect chymotrypsin to be most active at pH above 7. This is illustrated below with kinetic data collected on chymotrypsin.
Figure 3. The impact of pH variation on the kcat (catalytic constant) for chymotrypsin (left hand scale) and on the Km for an artificial substrate (high hand scale).
Figure from Zubay et al., Principles of Biochemistry, copyright ©1995 Wm. C. Brown Comm., Inc.
This diagram clearly shows that as the pH is raised from 6 the kcat increases until a pH of about 8 is reached, where His-57 would be fully de-protonated.
A second effect of pH on chymotrypsin activity results from a change in substrate binding. When chymotrypsin is activated by proteolysis in the intestine (as shown below in Fig. 4) Ile-16 becomes an additional 'N-terminal residue' and its free amino acid group helps position Asp-194 and the protein's backbone for substrate binding.
Figure 4. The proteolytic activation of chymotrypsin when the 'pro-enzyme' (called chymotrypsingen, which is inactive) is processed in the intestine. During activation of chymotryspin, two short sections of the amino acid backbone of the polypeptide are excised by tryptic and chymotryptic digestion (the latter being an example of self-digetion). However, the overall structure of chymotrypsin, in its activated forms which are called lambda-chymotrypsin (after tryptic digestion) and alpha-chymotrypsin (after the self digestion) is stabilized by two disulfide bonds which bridge the gaps in the backbone created by the removal of the short sections.
Figure from Voet's Biochemistry, copyright ©1990, John Wiley & Sons, Inc.
So titration of Ile-16 amino group (pK~9) leads to an increase in the Km at pH above 9 as shown in Figure 3 above. The Km increase means the substrate is more weakly bound at pH 9 and above. The net effect of pH on kcat and Km is that Chymotrypsin's pH optimum is 8, which is illustrated below with a plot of the specificity constant (ie kcat/Km) versus pH.
Figure 4A. Plot of kcat/Km (specificity constant) for chymotrypsin versus pH.
Figure from Zubay Biochemistry, 2nd Ed., 1988
Another example of pH effects at active site is found with ribonuclease (RNase) - an enzyme which degrades RNA. Why is ribonuclease most active at pH 6? Two His (His-12 and His-119) are involved in catalysis of RNA breakdown by RNase.
Figure 5. Model of a substrate molecule bound in the active site of RNase. The positions of His-12 and His-119 in the 3-D structure of RNase are shown in relation to the bond of the substrate to be hydrolyzed.
Figure from Zubay et al., Principles of Biochemistry, copyright ©1995 Wm. C. Brown Comm., Inc.
So it makes sense that RNase is most active at pH 6 since this is the pK of His. But one His must be protonated and one His unprotonated for the suggested mechanism. NMR expts have shown that one His has a pK of 5.4 and the other has a pK of 6.4.
Figure 6. pH optimum of ribonuclease.
Figure 7. Suggested Catalytic Mechanism of Ribonuclease.
Figure 6 & 7 from Voet's Biochemistry, copyright ©1990, John Wiley & Sons, Inc.
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©Wilbur H. Campbell, 1995; wcampbel@mtu.edu