Elastase substrate binding

FIGURE 16.19 The substrate-binding pockets of trypsin, chymotrypsin, and elastase. 

A straightforward approach is to hunt for short polypeptides that meet the specificity requirement of an enzyme but which, because of peculiarities of the sequence, are acted upon very slowly. Such a peptide may contain unusual or chemically modified amino acids. For example, the peptide Thr-Pro-nVal-NMeLeu-Tyr-Thr (nVal=norvaline NMeLeu = N-methylleucine) is a very slow elastase substrate whose binding can be studied by X-ray diffraction and NMR spectroscopy.6 Thiol proteases are inhibited by succinyl-Gln-Val-Val-Ala-Ala-p-nitroanilide, which includes a sequence common to a number of naturally occurring peptide inhibitors called cystatins.f They are found in various animal tissues where they inhibit cysteine proteases. 

Fig. 2. Schematic representation of the substrate-binding sites in the serine proteases (a) trypsin, (b) chymotrypsin and (c) elastase.

Within each protease family, individual members will differ in their substrate specificity. Most proteases have extended substrate binding sites and will bind to and recognize several amino acid residues of a polypeptide substrate (see Figure 2). Usually one of these will be the primary binding site. For example, in the serine proteases chymotrypsin, trypsin, and elastase, the primary substrate binding site is the Si subsite... 

Peptide chloromethyl ketone inhibitors have been developed for almost every serine protease that has been characterized adequately (30). For example, human leukocyte elastase, due to its involvement in emphysema, has been studied extensively with this class of inhibitor (32). The rate at which peptide chloromethyl ketones inhibit elastase is influenced by their interaction with the primary substrate binding site (Si) of the enzyme and by interactions at other subsites. The most effective chloromethyl ketone elastase inhibitor found thus far is MeO-Suc-Ala-Ala-Pro-ValCH2Cl (MeO-Suc- = CH3OCOCH2CH2CO-). This will not inhibit the other major leukocyte protease, cathepsin G (see Table VI). In contrast, Z-Gly-Leu-Phe-CH2C1 (Z = C6H5CH2OCO-) inhibits cathepsin G, but not elastase. Both enzymes can be inhibited with Ac-Ala-Ala-Pr o-V alCH2Cl. 

The protein superfamily of proteases [78, 79], however, is an ideal framework for a directed privileged structure-based masterkey concept. It has already been reported that the 5,5-trans-fused lactam moiety was systematically optimized and explored as a serine protease-directed scaffold by GlaxoSmithKline and has delivered progressible lead compounds for various members of that target class [3], such as thrombin [80, 81], elastase [82, 83], HCMV protease [84, 85], and the hepatitis C virus-encoded NS3-4A protease [86, 87]. Here, the initially identified scaffold was engineered toward the serine protease-wide commonality in substrate binding and processing [3],... 

Serine proteases are widely distributed and have many different functions. They are products of at least two evolutionary pathways, which originate in prokaryotes. Many of them resemble trypsin, chymotrypsin, elastase, or sub-tilisin in specificity, but serine proteases with quite different specificities have been isolated recently. A recent NMR study of a bacterial protease labelled with at carbon 2 of its single imidazole groups implicates a buried side chain of aspartic acid as the ultimate base for proton transfers in catalysis and eliminates a charge separation from reaction schemes for catalysis. Much of the catalytic effectiveness of serine proteases can be attributed to substrate binding, but the interactions which yield a Michaelis complex are supplemented by others which stabilize intermediates on the reaction pathway. 

Understanding how HLE inhibitors work and/or designing new inhibitors requires a model of HLE s active-site and an understanding of its mechanism of action. All serine proteinases share a similar catalytic region and mechanism of action but differ in several amino acids in the extended substrate-binding region. These changes are responsible for the specificity differences between HLE and other serine proteinases. In some cases analysis of the enzyme-inhibitor interactions has only been carried out with other related enzymes, and those results are referenced as appropriate. One closely related enzyme, porcine pancreatic elastase (PPE, EC 3.4.21.36) has... 

K. Nakajima, J. C. Powers, B. M. Ashe, and M. Zimmerman. Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the alpha 1-protease inhibitor reactive site. J. Biol. Chem. 254 4027 (1979). 

Such effects are not unique to carboxypeptidase A. The rate of substrate polysaccharide hydrolysis by lysozyme is remarkably dependent on the polysaccharide chain length 153, 154). Both steady-state kinetic studies and X-ray crystallographic studies on enzyme-inhibitor complexes for chymotrypsin 156) trypsin 156), elastase 157), and subtilisin 158) are indicative of the existence of multiple-loci substrate binding sites. Furthermore, the dependence of Acat on substrate chain length for all these enzymes strongly implies that the filling... 

Fig. 2.11. A hypothesis for substrate binding by a-chymotrypsin, trypsin and elastase enzymes (according to Shotton, 1971)...

Figure 2.3 Enzyme specificity — the substrate binding sites of trypsin, chymotrypsin and elastase.

This approach has been mainly applied to peptide-based inhibitors of proteases, where the inhibitory molecule is a peptide with a transition state isostere appended to it, and the cognate substrate is simply a peptide of the same amino acid sequence, but lacking the isostere functionality. Examples where good correlations between the free energy of inhibitor binding and the free energy of kcJKM have been found, include peptide-trifluoromethyl ketone inhibitors of human leukocyte elastase (Stein et al., 1987) and peptide-phosphonamidate inhibitors of the metalloprotease ther-molysin (Bartlett and Marlowe, 1983). 

Fig. 18. The active site region of the electron density difference map between N-carbobenzoxy-L-alanine-elastase at —SS C and native elastase at the same temperature. The resolution is 3.5 A. The bilobed feature is consistent with the binding of the alanyi portion of the substrate to the oxygen of the catalytic serine, with weak interaction of the carbobenzoxy group to the surface of the enzyme.

Effective concentration 65-72 entropy and 68-72 in general-acid-base catalysis 66 in nucleophilic catalysis 66 Elastase 26-30, 40 acylenzyme 27, 40 binding energies of subsites 356, 357 binding site 26-30 kinetic constants for peptide hydrolysis 357 specificity 27 Electrophiles 276 Electrophilic catalysis 61 metal ions 74-77 pyridoxal phosphate 79-82 Schiff bases 77-82 thiamine pyrophosphate 82-84 Electrostatic catalysis 61, 73, 74,498 Electrostatic effects on enzyme-substrate association rates 159-161... 

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