Serine protease, nucleophilicity

Structural analysis of the rhinovirus and the hepatitis A virus 3C proteases (Allaire et al. 1994 Matthews et al. 1994) confirmed earlier predictions that the picomavirus 3C proteases are similar to chymotrypsin-Uke serine proteases in their fold. An important difference is that the serine nucleophile of serine proteases is replaced with a cysteine however, the 3C protease is stracturally distinct from the eukaryotic cysteine protease class of enzymes. 

SCHEME 11.3 Postulated mechanisms for the inhibition of serine proteases by coumarin derivatives. NuH nucleophile. Pathway a suicide-type inactivation (suicide substrate). Pathway b transient inactivation by formation of a stable acyl-enzyme (alternate substrate-inhibitor). 

A practical enzymatic procedure using alcalase as biocatalyst has been developed for the synthesis of hydrophilic peptides.Alcalase is an industrial alkaline protease from Bacillus licheniformis produced by Novozymes that has been used as a detergent and for silk degumming. The major enzyme component of alcalase is the serine protease subtilisin Carlsberg, which is one of the fully characterized bacterial proteases. Alcalase has better stability and activity in polar organic solvents, such as alcohols, acetonitrile, dimethylformamide, etc., than other proteases. In addition, alcalase has wide specificity and both l- and o-amino acids that are accepted as nucleophiles at the p-1 subsite. Therefore, alcalase is a suitable biocatalyst to catalyse peptide bond formation in organic solvents under kinetic control without any racemization of the amino acids (Scheme 5.1). 

Cycloamyloses have been studied extensively as models of a-chymotrypsin and other serine proteases. Chymotypsin works via a double displacement pathway in which the hydroxyl group of serine-195 acts as a nucleophile. This is shown in Scheme I. In nature, the substrate is an amide. Synthetic esters are also used in model studies and routine assays. Nitrophenyl and... 

In chymotrypsin and other serine proteases the imidazole moiety of histidine acts as a general base not as a nucleophile as is probably the case in the catalysis of activated phenyl ester hydrolysis by (26). With this idea in mind, Kiefer et al. 40) studied the hydrolysis of 4-nitrocatechol sulfate in the presence of (26) since aryl sulfatase, the corresponding enzyme, has imidazole at the active center. Dramatic results were obtained. The substrate, nitrocatechol sulfate, is very stable in water at room temperature. Even the presence of 2M imidazole does not produce detectable hydrolysis. In contrast (26) cleaves the substrate at 20°C. Michaelis-Menten kinetics were obtained the second-order rate constant for catalysis by (26) is 10 times... 

Note that penicillins and structurally related antibiotics are frequently deactivated by the action of bacterial -lactamase enzymes. These enzymes also contain a serine residue in the active site, and this is the nucleophile that attacks and cleaves the P-lactam ring (see Box 7.20). The P-lactam (amide) linkage is hydrolysed, and then the inactivated penicillin derivative is released from the enzyme by further hydrolysis of the ester linkage, restoring the functional enzyme. The mode of action of these enzymes thus closely resembles that of the serine proteases there is further discussion in Box 7.20. 

Serine proteases, hydrolyzing a peptide bond Nucleophile 1st NH 2nd NH 3rdNH NH OH ... 

To summarize, the binding sites of lysozyme and the serine proteases are approximately complementary in structure to the structures of the substrates the nonpolar parts of the substrate match up with nonpolar side chains of the amino acids the hydrogen-bonding sites on the substrate bind to the backbone NH and CO groups of the protein and, for lysozyme, to the polar side chains of amino acids. The reactive part of the substrate is firmly held by this binding next to acidic, basic, or nucleophilic groups on the enzyme. 

In enzymes, the most common nucleophilic groups that are functional in catalysis are the serine hydroxyl—which occurs in the serine proteases, cholinesterases, esterases, lipases, and alkaline phosphatases—and the cysteine thiol—which occurs in the thiol proteases (papain, ficin, and bromelain), in glyceraldehyde 3-phosphate dehydrogenase, etc. The imidazole of histidine usually functions as an acid-base catalyst and enhances the nucleophilicity of hydroxyl and thiol groups, but it sometimes acts as a nucleophile with the phos-phoryl group in phosphate transfer (Table 2.5). 

Serine Proteases Enzymes that Use a Serine Residue for Nucleophilic Catalysis Ribonuclease A An Example of Concerted Acid-Base Catalysis... 

The serine proteases are a large family of proteolytic ( enzymes that use the reaction mechanism for nucleophilic catalysis outlined in equations (3) and (4), with a serine residue as the reactive nucleophile. The best known members of the family are three closely related digestive enzymes trypsin, chymotrypsin, and elastase. These enzymes are synthesized in the mammalian pancreas as inactive precursors termed zymogens. They are secreted into the small intestine, where they are activated by proteolytic cleavage in a manner discussed in chapter 9. 

Serine proteases. The ft- hydroxy group of serine acts as a nucleophile under slightly alkaline conditions examples are trypsin, chymotrypsin, and subtilisin. 

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