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 ...

Exogenous administration of 5-aminolevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of Protoporphyrin IX (PpIX) in photodynamic therapy. We analysed porphyrin formation in chemically induced squamous papillomas, after topical application of ALA and ALA hexyl ester (He-ALA) administered in different formulations, as well as the pattern of distribution in the internal organs, and the synthesis of porphyrins in distant tumoural and normal skins. A lotion formulation containing DMSO and ethanol was the best vehicle for topical ALA delivery to papillomas, whereas cream was the most efficient formulation for He-ALA application. Similar porphyrin concentration can be accumulated in the skin tumours employing either ALA or He-ALA delivered in their optimal formulations. The use of cream as a vehicle of both ALA and He-ALA, induces highest porphyrin tumour/normal skin ratios. The main advantage of using He-ALA is that porphyrins synthesized from the ester are more confined to the site of application, thus inducing low porphyrin levels in normal skin, liver, blood and spleen, as well as in papillomas distant from the point of application, independently on the vehicle employed, so reducing potential side effects of photodynamic therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com

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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.