PART IV THE CATALYTIC FUNCTION

Transcription

1 PAT IV TE ATALYTI FUNTIN

2 INTDUTIN The life of a cell, of an organism, depends on the multiplicity of the diverse chemical reactions which constitute metabolism. These reactions are carried out with appreciable rates in conditions where they could usually not occur, conditions of restricted temperature and near neutral p. They occur in these conditions because they are catalysed by protein catalysts, the enzymes. These are either globular proteins soluble in aqueous media or membrane proteins. Their activity depends very tightly on their three-dimensional structure and their internal dynamics; every modification of this structure, even very slight, can bring about variations important to the catalytic activity, indeed even the loss of it. Knowledge of the three-dimensional structure of an increasing number of enzymes constitutes precious data specifying the mechanism of their catalytic action. In addition, metabolic reactions implicate extremely subtle rules. ells, living beings are autoregulated. Enzymes are characterised both by a large catalytic efficiency and by their specificity. Taking into account the diversity of reactions that take place in living organisms, it is not surprising to find a great number of enzymes in cells. Enzymes are catalysts; like all catalysts, they act in very weak quantities and are regenerated at the end of the reaction. The efficiency of the action of an enzyme is often defined by the turn over number or molecular activity (see Part II). It is the number of substrate molecules that an enzyme molecule can transform per second. According to different enzymes, this number can vary between 10 2 and 10 8 s 1. Table I below gives an idea of the turn over number of several enzymes. It appears that enzymes of electron transfer including catalase and raifort peroxydase are among the most efficient. Enzymes of group transfer have weaker molecular activities. Nevertheless, this table shows the very great catalytic efficiency of enzymes which, of all the catalysts, are the most efficient. Enzymatic reactions proceed at rates much greater than reactions catalysed by chemical catalysts. If one compares for the same chemical reaction, when possible, the rate of the reaction catalysed by an enzyme and by a chemical catalyst, one can find efficiency factors 10 8 to times greater for the enzymatic catalysis than for the chemical catalysis.

3 344 MLEULA AND ELLULA ENZYMLGY Table 1 Molecular activity of selected enzymes Enzyme Molecular activity (s 1 ) atalase ytochrome oxydase arbonic anhydrase 3-ketosteroid isomerase aifort peroxidase Acetaldehyde reductase Acetylcholinesterase xaloacetate amino transferase Lactate dehydrogenase hymotrypsin Tryptophan synthase Lysozyme ne of the most remarkable characteristics of enzymatic catalysis compared to chemical catalysis is its specificity. The specificity of an enzyme is of two orders: the specificity for a reaction and the specificity with regard to the substrate. Each enzyme can only catalyse a single reaction or one type of reaction; it has a well defined function. For example, a protease catalyses the hydrolysis of peptide bonds, a dehydrogenase a dehydrogenation reaction, a glycosidase the breaking of glycosidic bonds etc. Each enzymatic protein generally supports a single function. There exist however multifunctional enzymes, but these are generally constituted of the assembly of several subunits, each carrying a defined function, or are proteins comprised of several structural domains, each function relevant to a distinct domain (see Part V). In addition, each enzyme recognises a well determined substrate or type of substrate. The required conditions for a compound to be a substrate of an enzyme can be more or less strict. Diverse enzymes have more or less large specificities. Several examples will be given. All pancreatic proteases catalyse the hydrolysis of peptide bonds, but trypsin attacks the bonds for which the carboxyl belonging to an L-lysine or an L-arginine, whereas chymotrypsin requires the presence of aromatic amino acids. Figure 1 opposite illustrates the difference in specificity of these two proteases. In addition, these are only active with respect to amino acids in the L configuration. ptic isomers of the D configuration are not substrates of these enzymes; however, they are recognised and can be bound to the active site with affinities comparable to those of their L isomers, but the peptide bonds are not (or are very slowly) hydrolysed. Enzymes are therefore stereospecific. utside of this condition, the specificity of these enzymes is not absolutely strict; each of these proteases is capable of hydrolysing the substrates of the other, but with a much lower efficiency.

4 INTDUTIN 345 Substrates of trypsin: N X ( ) 4 ( ) 3 N 3 + N Substrates of chymotrypsin: + N 3 N Fig. 1 Specificity of trypsin and chymotrypsin X = N N peptide X = N amide X = ester Galactosidases are enzymes presenting a much stricter specificity. β-galactosidase hydrolyses β-galactosides (β-d-galactopyranosides), but not α-galactosides. onversely, α-galactosidase hydrolyses α-galactosides but remains without action on β-galactosides. A very precise configuration is required so that the respective compounds are substrates of these enzymes. The inversion or the modification of one of the hydroxyls of the ring of β-galactosides suffices so that the compound is no longer a substrate of β-galactosidase; thus 2-methyl-β-D-galactosides, or β-d-glucosides resulting in the inversion of the position of the hydroxyl in 4, are no longer substrates of β-galactosidase. β-d-fucosides are poor substrates due to the replacement of the hydroxyl in 6 by a single proton. The replacement of oxygen in the glycosidic bond by a sulfur to form a β-d-thiogalactoside gives a compound that is not hydrolysed by β-galactosidase; it behaves as a competitive inhibitor (Fig. 2 below). ne of the interests of enzyme catalysis resides in the ability to distinguish between forms of homologous compounds when it is often impossible to separate them by chemical means. In particular, one must underline the importance of the specificity of enzymes in relation to enantiomorphic compounds. The enzyme generally only attacks one of the two optic isomers, whereas asymmetric syntheses are very difficult to carry out by chemical methods. PASTEU succeeded in separating L and D tartric acids from a racemic mixture by using a mold which transformed only L-tartrate because it contained an enzyme, L-tartrate dehydratase. ne knows today that there exists a L- and a D-tartrate dehydratase which hydrolyses respectively each of the two optic isomers with the exclusion of the other (Fig. 3 below).

5 346 MLEULA AND ELLULA ENZYMLGY -D-galactoside -D-galactoside 3 2-methyl- -D-galactoside -D-glucoside 3 S -D-fucoside -D-thiogalactoside Fig. 2 Specificity of galactosidases L-tartrate D-tartrate 2 2 xaloacetic acid Fig. 3 Separation of L and D-tartrate by the corresponding enzymes It is important to understand by which mechanisms enzymes, protein macromolecules, are the most efficient catalysts that exist and at the same time the most specific. The diverse factors that can account for this are analysed in this part in light of the most recent data.