Serine relative hydrophobicity

As stated earlier, lipases act at the interface between hydrophobic and hydrophilic regions, a characteristic that distinguishes lipases from esterases. Similar to serine proteases, lipases share the nucleophile-histidine-acidic residue catalytic triad that manifests itself as either a Ser-His-Asp triad or a Ser-His-Glu triad. The enzyme s catalytic site often is buried within the protein structure, surrounded by relatively hydrophobic residues. An a-helical polypeptide structure acts as a cover, making the site inaccessible to solvents and substrates. For the lipase to be active, the a-helical lid structure has to open so that the active site is accessible to the substrate. The phenomenon of interfacial activation is often associated with reorientation of the lid, increasing the hydrophobicity of the surface in the vicinity of the active site and exposing it. The opening of the lid structure may be initiated on interaction with an oiFwater interface. 

Figure 9.10 Specificity pocket of chymotrypsin. Notice that this pocket is lined with relatively hydrophobic residues and is relatively deep, favoring the binding of residues with long hydrophobic side chains such as phenylalanine (shown in green). Also notice that the active-site serine residue (serine 195) is positioned to cleave the peptide backbone between the residue bound in the pocket and the next residue in the sequence. The key amino acids that constitute the binding site are labeled.

The actual reaction mechanism is very similar for the different members of the family, but the specificity toward the different side chain, R, differs most strikingly. For example, trypsin cleaves bonds only after positively charged Lys or Arg residues, while chymotrypsin cleaves bonds after large hydrophobic residues. The specificity of serine proteases is usually designated by labeling the residues relative to the peptide bond that is being cleaved, using the notation... 

When switching from water to an organic solvent, or switching between organic solvents, the substrate specificity can change. In the example of the standard reaction, transesterification of N-acetyl-i-phenylalanine ethyl ester with n-propanol by Subtilisin Carlsberg, which has been mentioned several times in this chapter already, the relative specificity between the rather hydrophobic phenylalanine compound and its more hydrophilic analog N-acetyl-L-serine ethyl ester varies with the solvent (Table 12.8) (Wescott, 1993). 

Murakami et al. utilized catalytic bilayer membranes to catalyze the (1-replacement reaction of serine with indoles [44], The bilayer vesicle formed with 32 and 36 drastically accelerated the (1-replacement reaction by 51-fold (krel) relative to pyridoxal in homogeneous aqueous solution. They attributed this to the hydrophobic microenvironmental effect provided by the bilayer vesicle, which affords effective incorporation of indole molecules and elimination of water molecules in the reaction site. The imida-zolyl group of 33 enhanced the reaction further, krd being 130, possibly due to general acid-base catalysis by the imidazolyl group. Copper(n) ions also improved the reaction. 

In the recent studies, the enzyme shows that the overall polypeptide fold of chymotrypsin-like serine protease possesses essential SI specificity determinants characteristic of elastase using the multiple isomorphous replacement (MIR) method and refined to 2.3 A resolution Fig. (5). Structure-based inhibitor modeling demonstrated that EFEa s SI specificity pocket is preferable for elastase-specific small hydrophobic PI residues, while its accommodation of long and/or bulky PI residues is also feasible if enhanced binding of the substrate and induced fit of the SI pocket are achieved [Fig. (6) shows the active sites of serine protease]. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. This structure is the first report of an earthworm fibrinolytic enzyme component, a serine protease originated from annelid worm [17]. 

The possibility of HjO as the hydrogen donor is unlikely, because D is in all likelihood buried inside the strongly hydrophobic, membrane-spanning a-helix region of the Dj protein subunit. We therefore suggest that the proton donating species is an amino acid. The folding of the Dj polypeptide proposed in [14] results in four possiblities hisidine or serine in the second, or tryptophan or serine in the fifth intramembrane a-helix. A selection between these donors can only be made when a more precise location of the different a-helices relative to the tyrosyl donor is known. 

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