Enzymes ionization

The pH-dependence of the inhibition also indicated that unprotonated castanospermine is a better inhibitor than the protonated form. However, as essential carboxyl groups of the enzyme ionize in the same range of pH as castanospermine (pK, 6.09), it was not possible to estimate the inhibitory potency of protonated castanospermine. 

The simplest Michaelis-Menten-type scheme for showing analytically the effect of enzyme ionization on enzyme kinetic parameters is... 

It is often the case that the substrate has a required protonation state for binding and/or catalysis, and, similarly, catalytic groups in the active site usually have required protonation states as well. Thus, when the substrate or a key group on the enzyme ionizes in the accessible pH range, there will be a variation of the kinetic parameters with pH. Such pH variation often provides a clue to the acid-base catalysis, and thus the chemical mechanism of the reaction. The interested reader may find other articles on pH profiles useful (2, 3, 77). 

As noted above, pH profiles are plotted as log-log plots, and they consist of linear segments with whole number slopes connected by curved segments 2 pH units wide. We consider first pAj profiles for a competitive inhibitor (or substrate that adds prior to the last substrate). This profile shows only the pA values of the inhibitor (or substrate) or of groups on the enzyme that are important for binding. When only the deprotonated inhibitor will bind and no groups on the enzyme ionize, the pAt profile will be flat from 1 pH unit above the pA of the inhibitor to the high-pH end of the profile, and it will have a slope of 1 at low pH ... 

The use of mass spectrometry for the analysis of peptides, proteins, and enzymes has been summarized. This chapter should be read in conjunction with others, including Chapter 45, An Introduction to Biotechnology, and Chapters 1 through 5, which describe specific ionization techniques in detail. 

FIGURE 14.11 The pH activity profiles of four different enzymes. Trypsin, an intestinal protease, has a slightly alkaline pH optimnm, whereas pepsin, a gastric protease, acts in the acidic confines of the stomach and has a pH optimmn near 2. Papain, a protease found in papaya, is relatively insensitive to pHs between 4 and 8. Cholinesterase activity is pH-sensitive below pH 7 but not between pH 7 and 10. The cholinesterase pH activity profile suggests that an ionizable group with a pK near 6 is essential to its activity. Might it be a histidine residue within the active site ... 

The enzyme carbonic anhydrase promotes the hydration of COg. Many of the protons formed upon ionization of carbonic acid are picked up by Hb as Og dissociates. The bicarbonate ions are transported with the blood back to the lungs. When Hb becomes oxygenated again in the lungs, H is released and reacts with HCO3 to re-form HgCOj, from which COg is liberated. The COg is then exhaled as a gas. 

The first hint that two active-site carboxyl groups—one proto-nated and one ionized—might be involved in the catalytic activity of the aspartic proteases came from studies of the pH dependence of enzymatic activity. If an ionizable group in an enzyme active site is essential for activity, a plot of enzyme activity versus pH may look like one of the plots at right. 

Bell-shaped activity versus pH profiles arise from two separate active-site ionizations, (a) Enzyme activity increases upon deprotonation of (b) Enzyme activity decreases upon deprotonation of A-H. (c) Enzyme activity is maximal in the pH range where one ionizable group is deprotonated (as B ) and the odier group is protonated (as A-H). 

Peptide mass fingeiprinting (PMF) is a mass spectrometry based method for protein identification. The protein is cleaved by an enzyme with high specificity (trypsin, Lys-C, Asp-N, etc.) or chemical (CNBr). The peptide mixture generated is analyzed by matrix-assisted laser desorp-tion/ionization (MALDI) or electrospray ionization (ESI)... 

This calculation demonstrates that a nonpolar solvent can accelerate S 2 reactions. However, this is not what we are asking the relevant quantity is the overall activation energy for the reaction in a nonpolar enzyme which is surrounded by water. Thus, as is indicated in the thermodynamic cycle of Fig. 9.3, we should include the energy of moving the ionized R-O- from water to the nonpolar active site (AAg j1). Thus the actual apparent change in activation barrier is... 

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