BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

PART I. CONFORMATION AND DYNAMICS

CHAPTER 7. MECHANISM OF ENZYME ACTION: LYSOZYME AND CARBOXYPEPTIDASE

Summary

Lysozyme is a relatively small enzyme that cleaves the polysaccharide component of bacterial Cell walls. Structurally, this polysaccharide is an alternating polymer of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) residues, linked by β(l → 4) glycosidic bonds. Lysozyme hydrolyzes the glycosidic bond between C-1 of the NAM residue and C-4 of the NAG residue. Oligomers of N-acetylglucosamine are also hydrolyzed by lysozyme. Specifically, hexa-NAG and longer polymers are readily cleaved by the enzyme, whereas tri-NAG and di-NAG are hydrolyzed at an extremely low rate. Tri-NAG is a potent competitive inhibitor of the enzyme. The three-dimensional Structure of lysozyme and its complex with tri-NAG has been solved at atomic resolution. It has been established that tri-NAG occupies half of the cleft running across the enzyme molecule, binding to it through numerous Hydrogen Bonds and Van der Waals interactions. Based on the structural data of the lysozyme-tri-NAG complex, a model was constructed to predict how its active substrate, hexa-NAG, binds to lysozyme.

A hypothesis for The Mechanism of lysozyme catalysis has been proposed; its essence is as follows. First, the critical groups for catalysis are the un-ionized carboxylic acid group of the glutamate-35 residue and the carboxylate ion of aspartate-52. Both groups are located about 3 A from the glycosidic bond being hydrolyzed, namely the bond between residues D and E of the hexameric substrate. Second, glutamate-35 donates an H⁺ to the glycosidic bond between C-1 of ring D and the glycosidic oxygen atom, leading to the Cleavage of this bond. C-1 of ring D becomes positively charged; this Transition State is called a carbonium ion. Third, the carbonium ion reacts with an OH ^- group from the solvent, while glutamate-35 accepts a proton, returning to its original protonated state. Once the reaction products diffuse away from the enzyme, lysozyme is ready for a new catalytic cycle. Fourth, The rate of catalysis is significantly enhanced by two factors that facilitate The intermediate formation of the carbonium ion: an electrostatic factor, namely the proximity of the negatively charged aspartate-52 side chain, and a geometric factor, in which ring D is distorted into a half-chair conformation, which distributes the positive charge of the carbonium ion between C-1 and the oxygen atom of the carbohydrate ring.

Carboxypeptidase A, a digestive enzyme that cleaves the C-terminal peptide bond in Polypeptides, exemplifies an enzyme operating by a completely different catalytic mechanism. The structure of this enzyme and its complex with glycyltyrosine—a substrate analog—has been solved at atomic resolution. The binding of glycyltyrosine induces large structural Changes in the Active Site region, resulting in the exclusion of Water and making this region hydrophobic. The example of carboxypeptidase A illustrates the important role that induced fit plays in catalysis. Another striking feature of this enzyme is that its active site contains a zinc ion, which is essential for catalysis. The carbonyl carbon atom of the peptide bond to be cleaved is polarized by the zinc ion, making it more susceptible to nucleophilic attack. Here we see an example of induced electronic strain in the substrate. During catalysis by carboxypeptidase A, a water molecule activated by glutamate-270 directly attacks the carbonyl group of the peptide bond being cleaved; simultaneously, Tyrosine-248 donates a proton to the NH group of this bond, resulting in Hydrolysis.