2.1 How enzymes work: Active sites and catalytic mechanisms

Open any textbook of biochemistry and you will be presented with an overwhelming number of figures depicting the crystal structures of a multitude of enzymes. Of course, the three-dimensional structures of enzymes are crucial to their functions. Many enzymes are just regular protein molecules, composed of nothing else than the 20 standard amino acids. If you mix of all these amino acids in free form at, say, 10 mM each, this mixture will not have any significant catalytic activity. It therefore is the precise arrangement of the amino acids in the enzyme molecule that brings about the function.

As the α-amino and α-carboxyl groups of the amino acids are hooked up to each other in the polypeptide chain, they usually do not directly contribute to the catalytic effect of the enzyme. Instead, it is the side chains that are directly engaged win the reaction¹. A very good example of this is chymotrypsin . Chymotrypsin is one of the major proteases in the human digestive tract, where its job is to knock down large protein molecules into small peptides that are then further processed by peptidases.

How does chymotrypsin do that? The enzyme-catalyzed reaction (Figure 2.1-1c) is similar to alkaline hydrolysis (Figure 2.1-1d). In alkaline hydrolysis, a hydroxide ion, which is a strong nucleophile, attacks the carbon in the peptide bond that carries a partial positive charge. In the enzyme-catalyzed reaction, a deprotonated serine residue of the enzyme (Ser 95) plays a role similar to that of the hydroxide ion.

Now, you know that serine normally is a neutral amino acid – its OH-group does not spontaneously dissociate, no more than the -OH group of alcohol does². The question therefore is, how does the enzyme deprotonate its own serine side chain? This is brought about by placing the serine next to a histidine and an aspartate inside the active center (Figure 2.1-1b). Aspartate deprotonates histidine, which in turn deprotonates the serine residue. This motive – asp, his, ser – is very widespread among proteases and esterases, so much so that it is commonly called 'the catalytic triad'. E.g., the protease trypsin and several lipases that occur in human metabolism have this motif and share the same mechanism of catalysis.

While with many enzymes the protein molecule and its amino acid side chains are sufficient for catalysis, many others require co-enzymes for their catalytic activity. Very often, both active-site amino acid side chains and coenzymes are required. For example, amino acid transaminases (cf. Figure 12.2-2) have a molecule of the coenzyme pyridoxalphosphate bound to the active site, which co-operates with an active site lysine in the enzyme's catalytic activity.

Most enzymes have just one active site, or if they are multimeric one active site per subunit. However, there are exceptions: Fatty acid synthase (cf. section 10.4) has as many as eight different active sites on each subunit. Multi-enzyme complexes such as pyruvate dehydrogenase (cf. section 5.1) have one active site per subunit but combine different types of subunits and enzyme activities in one functional assembly.

1: You will note that, nevertheless, in the textbooks many enzyme structures are just given as ribbon diagrams that show the fold of the protein backbone but omit the side chains. Therefore, these diagrams are quite useless in terms of understanding the catalytic mechanism of an enzyme.

2: Otherwise, beer would taste sour – how rotten that would be!