Serine carbonyl oxygen

X-ray crystallographic studies of serine protease complexes with transition-state analogs have shown how chymotrypsin stabilizes the tetrahedral oxyanion transition states (structures (c) and (g) in Figure 16.24) of the protease reaction. The amide nitrogens of Ser and Gly form an oxyanion hole in which the substrate carbonyl oxygen is hydrogen-bonded to the amide N-H groups. 

From the first transition state (TSl, Fig. 1), the reaction path leads to the tetrahedral intermediate 1 (INTI). In the latter, the proton transfer from methanol to the tertiary amine function is completed (from 1.183 to 1.059 A), and the negative charge at the former carbonyl oxygen atom reaches its maximum. This charge is compensated by a further shortening of the bifurcated hydrogen bonds to 2.040 A (-0.103 A) and 1.765 A (-0.096 A) (Fig. 1). The thiourea moiety thus forms an oxyanion hole similar to the amide groups of the serine protease backbone [41]. 

The first crystal structure of a bacterial serine protease to be solved—subtilisin, from Bacillus amyloliquefaciens—revealed an enzyme of apparently totally different construction from the mammalian serine proteases (Figure 1.17). This was not unexpected, since there is no sequence homology between them. But closer examination shows that they are functionally identical in terms of substrate binding and catalysis. Subtilisin has the same catalytic triad, the same system of hydrogen bonds for binding the carbonyl oxygen and the acetamido NH of the substrate, and the same series of subsites for binding the acyl portion of... 

Local interactions between side chains and main-chain C=0 groups are dominated by serine and threonine in a helices and (less often) in turns. These residues usually make a second hydrogen bond to a carbonyl oxygen atom already bonded to a main-chain amide. 

The probe then has to spin about an imaginary sp2-sp2 axis (A in Fig. 1.3) which links it to the backbone carbonyl oxygen, until the probe s own lone pair points as directly as possible towards the sp3 hydroxy oxygen of the serine. 

The proposed catalytic mechanism involves a proton relay-type reaction where an immobilized HjO molecule serves as the nucleophile, a role normally filled by serine. The proton relay system is buried in the hydrophobic active-site wall. Ca " " binds the phosphate moiety of the substrate and, serving as a Lewis acid, polarizes the ester bond at the carbonyl oxygen. The HjO molecule, immobilized by the Asp-His pair attacks the carbonyl of the substrate and donates an H" " to His. The alkoxy oxygen of the glycerol backbone then retrieves the H" " from the His to complete the reaction. 

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