Histidine acid-base catalysis

The cleavage mechanism of the caspases is shown schematically in Fig. 15.5. They use a typical protease mechanism with a catalytic diad for cleavage of the peptide bond. The nucleophilic thiol of an essential Cys residue forms a covalent thioacyl bond to the substrate during the catalysis. The imidazole ring of an essential histidine is also involved in catalysis and this facilitates hydrolysis of the amide bond in the sense of an acid/base catalysis. 

The pH-rate profile for the action of the enzyme shows a typical pH maximum, with sharply lower rates at either higher or lower pH than the optimum these facts suggest that both an acidic and a basic group are required for activity (Herries, 1960). The two essential histidine residues could serve as these groups if, in the active site, one were protonated and the other present in its basic form. The simultaneous acid-base catalysis would parallel that of the model system (discussed below) of Swain and J. F. Brown. The essential lysine, which binds phosphate, presumably serves to bind a phosphate residue of the ribonucleic acid. These facts led Mathias and coworkers to propose the mechanism for the action of ribonuclease that is shown in (13) (Findlay et al., 1961). 

Bronsted acid/base catalysis is the most common enzymatic mechanism, since nearly all enzymatic reactions involve a proton transfer. This means that nearly all enzymes have acidic and/or basic groups in their active site. In add catalysis, the substrate is protonated by one of the amino add residues at the active site (typically aspartic acid, glutamic acid, histidine, cysteine, lysine, or tyrosine). This residue itself must therefore be protonated at the readion pH (typically between pH 5 and 9), with a pKa just above this value. Conversely, in base catalysis, the pJCa of the deprotonating residue must be just below the physiological pH. Some enzymes can even carry out bifunctional catalysis, by protonating and deprotonating two different sites on the same substrate molecule simultaneously. 

Ribonuclease A is a member of a group of enzymes that cleave RNA using general acid-base catalysis without a metal ion in the enzyme. In ribonuclease A, such catalysis is performed by two imidazoles of histidine units, one as the free base (Im) and the other, protonated, as the acid (ImH+). To mimic this in an artificial enzyme, we prepared (3-cyclodextrin bis-imidazoles 41 [124]. The first one was a mixture of the... 

Acid-base catalysis does not contribute to rate enhancement by a factor greater than —100, but together with other mechanisms that operate in the active site of an enzyme, it contributes considerably to increasing the enzymatic rate of reactions. The amino acid side chains of glutamic acid, histidine, aspartic acid, lysine, tyrosine, and cysteine in their protonated forms can act as acid catalysts and in their unprotonated forms as base catalysts (see Prob. 8.11). Clearly, the effectiveness of the side chain as a catalyst will depend on the p/ffl (Chap. 3) in the environment of the active site and on the pH at which the enzyme operates. 

General acid-base catalysis. In general acid-base catalysis, a molecule other than water plays the role of a proton donor or acceptor. Chymotrypsin uses a histidine residue as a base catalyst to enhance the nucleophilic power of serine (Section 9.1.3). 

Figure 9.8. Peptide Hydrolysis by Chymotrypsin. The mechanism of peptide hydrolysis illustrates the principles of covalent and acid-base catalysis. The dashed green lines indicate favorable interactions between the negatively charged aspartate residue and the positively charged histidine residue, which make the histidine residue a more powerful base.

The molecular components of many buffers are too large to reach the active site of carbonic anhydrase. Carbonic anhydrase II has evolved a proton shuttle to allow buffer components to participate in the reaction from solution. The primary component of this shuttle is histidine 64. This residue transfers protons from the zinc-bound water molecule to the protein surface and then to the buffer (Figure 9.30). Thus, catalytic function has been enhanced through the evolution of an apparatus for controlling proton transfer from and to the active site. Because protons participate in many biochemical reactions, the manipulation of the proton inventory within active sites is crucial to the function of many enzymes and explains the prominence of acid-base catalysis. 

Next, we come to the question what is the role of histidine-57 We are observing an example of general acid-base catalysis catalysis not just by hydroxide ions and oxonium ions, but by all the bases and conjugate acids that are present, each contributing according to its concentration and its acid or base strength. 

Figure 37.2 depicts the action of chymolrypsin, with the imidazole group of histidine-57 playing the same role of general base as that just described—and with protonated imidazole necessarily acting as general acid. There is general acid-base catalysis of both reactions involved first, in the formation of the acyl enzyme, and then in its hydrolysis. 

Usually acid/base catalysis is provided by the amino acid histidine. Histidine is a weak base and can easily equilibrate between its protonated form and its free base form (Fig. 4.19). In doing so, it can act as a proton bank that is, it has the capability to accept and donate protons in the reaction mechanism. This is important since active sites are frequently hydrophobic and will therefore have a low concentration of water and an even lower concentration of protons. 

The histidine residue acts as an acid/base catalyst throughout the mechanism, while serine plays the part of a nucleophile. This is not a particularly good role for serine since an aliphatic alcohol is a poor nucleophile. In fact, serine by itself is unable to hydrolyse an ester. However, the fact that histidine is close by to provide acid/base catalysis overcomes that disadvantage. There are several stages to the mechanism. 

The main parts of this scheme were proposed earlier by Theorell and co-workers 119,291) on the basis of inhibitor binding and steady-state kinetic studies. Other suggested mechanisms based on general acid-base catalysis 297), reduction of the enzyme 362), or direct participation of histidine 363) or cysteine 364) in the hydride transfer step are highly unlikely in view of the crystallographic and kinetic results reviewed in this chapter. Contrary to expectations the mechanism described here is in most details very different from that proposed for lactic dehydrogenase 126). 

The kinetics of PAPs exhibit a bell-shaped pH—rate dependency, typical of acid—base catalysis. NMR data obtained with recombinant human PAP between pH 5.5 and pH 7.1 indicates that pA 2 does not involve a metal ligand, and may instead be due to the ionization of one of the two conserved histidine residues near the active site. It has been proposed that one of these histidine residue acts as a general acid in protonation of the leaving group. ° The other histidine residue (H92 in the human PAP and H202 in the kidney bean PAP ) has been suggested to assist in substrate positioning. 

To participate in general acid-base catalysis, the amino acid side chain must be able to abstract a proton at one stage of the reaction, and donate it back at another. Histidine (pK, 6.3) would be pro-tonated at this low pH, and could not abstract a proton from a potential nucleophile. However, aspartic acid, with a (pK of about 2) can release protons at a pH of 2. The two aspartates work together to activate water through the removal of a proton to form the hydroxyl nucleophile. 

C) inhibit intestinal enzymes dependent on histidine for acid-base catalysis. 

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