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Benzyloxycarbonyl (Z)-Ala-Pro-Phe-glyoxal and Z-Ala-AlaPhe-glyoxal have both been shown to be inhibitors of ␣-chymotrypsin with minimal K i values of 19 and 344 nM, respectively, at neutral pH. These K i values increased at low and high pH with pK a values of ϳ4.0 and ϳ10.5, respectively. By using surface plasmon resonance, we show that the apparent association rate constant for Z-Ala-Pro-Phe-glyoxal is much lower than the value expected for a diffusion-controlled reaction.13 C NMR has been used to show that at low pH the glyoxal keto carbon is sp 3 -hybridized with a chemical shift of ϳ100.7 ppm and that the aldehyde carbon is hydrated with a chemical shift of ϳ91.6 ppm. The signal at ϳ100.7 ppm is assigned to the hemiketal formed between the hydroxy group of serine 195 and the keto carbon of the glyoxal. In a slow exchange process controlled by a pK a of ϳ4.5, the aldehyde carbon dehydrates to give a signal at ϳ205.5 ppm and the hemiketal forms an oxyanion at ϳ107.0 ppm. At higher pH, the re-hydration of the glyoxal aldehyde carbon leads to the signal at 107 ppm being replaced by a signal at 104 ppm (pK a ϳ9.2). On binding either Z-Ala-Pro-Phe-glyoxal or Z-AlaAla-Phe-glyoxal to ␣-chymotrypsin at 4 and 25°C, 1 H NMR is used to show that the binding of these glyoxal inhibitors raises the pK a value of the imidazolium ion of histidine 57 to a value of >11 at both 4 and 25°C. We discuss the mechanistic significance of these results, and we propose that it is ligand binding that raises the pK a value of the imidazolium ring of histidine 57 allowing it to enhance the nucleophilicity of the hydroxy group of the active site serine 195 and lower the pK a value of the oxyanion forming a zwitterionic tetrahedral intermediate during catalysis.Specific substrate-derived glyoxal inhibitors have been shown to be potent inhibitors of the serine proteinases (1-4). Z 4 -Ala-Pro-Phe-glyoxal is an extremely potent reversible inhibitor of ␦-chymotrypsin with an apparent disassociation constant of 25 Ϯ 8 nM at pH 7.0 (1).The ␣-keto carbon of the glyoxal inhibitor is expected to occupy the same position as the carbonyl carbon of a substrate, and it has been shown that it is bound as a tetrahedral adduct, which should closely resemble the tetrahedral intermediate formed during substrate catalysis (1). By using 13 C NMR, it has been shown that ␦-chymotrypsin (1) and subtilisin (2) reduce the oxyanion pK a by ϳ6 and ϳ8 pK a units, respectively. It has been estimated that hydrogen bonding in the oxyanion hole will only reduce the oxyanion pK a by ϳ1.3 pK a units (1). This is consistent with the fact that hydrogen bonding is expected to be effective in both water and in the oxyanion hole, and so it should not reduce the oxyanion pK a to a value lower than that expected in water. This has led to the conclusion that hydrogen bonding in the oxyanion hole only has a minor role in lowering the oxyanion pK a (5-7). However, it has been proposed that substrate binding raises the pK a of the imidazolium ion of the active site histidine enabling ...

A high intrapulmonary protease burden is characteristic of cystic fibrosis (CF), and the resulting dysregulation of the protease/anti-protease balance has serious implications for inflammation in the CF lung. Because of this inflammation, microbleeds can occur releasing hemoglobin into the lung. The aim of this study was to investigate the effect of the protease-rich environment of the CF lung on human hemoglobin and to assess the proinflammatory effect of heme on CF bronchial epithelium. Here, we show that the Pseudomonas proteases (Pseudomonas elastase and alkaline protease) and the neutrophil proteases (neutrophil elastase (NE) and proteinase-3) are capable of almost complete degradation of hemoglobin in vitro but that NE is the predominant protease that cleaves hemoglobin in vivo in CF bronchoalveolar lavage fluid. One of the effects of this is the release of heme, and in this study we show that heme stimulates IL-8 and IL-10 protein production from ⌬F508 CFBE41o ؊ bronchial epithelial cells. In addition, heme-induced IL-8 expression utilizes a novel pathway involving meprin, EGF receptor, and MyD88. Meprin levels are elevated in CF cell lines and bronchial brushings, thus adding to the proinflammatory milieu. Interestingly, ␣ 1 -antitrypsin, in addition to its ability to neutralize NE and protease-3, can also bind heme and neutralize heme-induced IL-8 from CFBE41o؊ cells. This study illustrates the proinflammatory effects of micro-bleeds in the CF lung, the process by which this occurs, and a potential therapeutic intervention.Cystic fibrosis (CF) 2 is a chronic inflammatory disease, which is associated with high protease levels in the lung. The normal fine balance between the positive physiological function of the proteases and their deleterious effect is dysregulated in the CF lung. This results in unopposed protease activity that can cause lung damage.In the CF lung, proteases are secreted by the host and also by bacteria that colonize the lung. The host secretes serine, metallo, cysteine, and aspartyl proteases that include elastases, matrix metalloproteases, cathepsins, prolyl endopeptidases, and napsins. However, it is the elastases that are the main contributor to the total protease activity found in the CF lung. Given the abundance of neutrophils in CF, the principal lung proteases are secreted by the neutrophil. These include neutrophil elastase (NE), protease-3 (PR-3), and cathepsin G. NE is thought to contribute 90% of the CF sputum elastinolytic activity (1) with PR-3 contributing 7% and the balance of activity derived from the remaining proteases. NE cleaves a broad spectrum of proteins, including immunoglobulins, plasma proteins, matrix proteins, cytokines, and protease inhibitors. In addition, NE can amplify inflammatory signaling by direct and transcriptional (2) regulation or activation of other proteases and induction of IL-8 from airway epithelium (3-5).Although often disregarded, bacteria within the lung also contribute to the protease load in the CF lung. The range of bacteria that colo...

Z-Ala-Ala-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of pepsin with a Ki = 89 +/- 24 nM at pH 2.0 and 25 degrees C. Both the ketone carbon (R13COCHO) and the aldehyde carbon (RCO13CHO) of the glyoxal group of Z-Ala-Ala-Phe-glyoxal have been 13C-enriched. Using 13C NMR, it has been shown that when the inhibitor is bound to pepsin, the glyoxal keto and aldehyde carbons give signals at 98.8 and 90.9 ppm, respectively. This demonstrates that pepsin binds and preferentially stabilizes the fully hydrated form of the glyoxal inhibitor Z-Ala-Ala-Phe-glyoxal. From 13C NMR pH studies with glyoxal inhibitor, we obtain no evidence for its hemiketal or hemiacetal hydroxyl groups ionizing to give oxyanions. We conclude that if an oxyanion is formed its pKa must be >8.0. Using 1H NMR, we observe four hydrogen bonds in free pepsin and in pepsin/Z-Ala-Ala-Phe-glyoxal complexes. In the pepsin/pepstatin complex an additional hydrogen bond is formed. We examine the effect of pH on hydrogen bond formation, but we do not find any evidence for low-barrier hydrogen bond formation in the inhibitor complexes. We conclude that the primary role of hydrogen bonding to catalytic tetrahedral intermediates in the aspartyl proteases is to correctly orientate the tetrahedral intermediate for catalysis.