1. Hemiacetal stabilization in a chymotrypsin inhibitor complex and the reactivity of the hydroxyl group of the catalytic serine residue of chymotrypsin
Jennifer A Cleary, William Doherty, Paul Evans, J Paul G Malthouse Biochim Biophys Acta. 2014 Jun;1844(6):1119-27. doi: 10.1016/j.bbapap.2014.03.008. Epub 2014 Mar 21.
The aldehyde inhibitor Z-Ala-Ala-Phe-CHO has been synthesized and shown by (13)C-NMR to react with the active site serine hydroxyl group of alpha-chymotrypsin to form two diastereomeric hemiacetals. For both hemiacetals oxyanion formation occurs with a pKa value of ~7 showing that chymotrypsin reduces the oxyanion pKa values by ~5.6 pKa units and stabilizes the oxyanions of both diastereoisomers by ~32kJmol(-1). As pH has only a small effect on binding we conclude that oxyanion formation does not have a significant effect on binding the aldehyde inhibitor. By comparing the binding of Z-Ala-Ala-Phe-CHO with that of Z-Ala-Ala-Phe-H we estimate that the aldehyde group increases binding ~100 fold. At pH7.2 the effective molarity of the active site serine hydroxy group is ~6000 which is ~7× less effective than with the corresponding glyoxal inhibitor. Using (1)H-NMR we have shown that at both 4 and 25°C the histidine pKa is ~7.3 in free chymotrypsin and it is raised to ~8 when Z-Ala-Ala-Phe-CHO is bound. We conclude that oxyanion formation only has a minor role in raising the histidine pKa and that the aldehyde hydrogen must be replaced by a larger group to raise the histidine pKa>10 and give stereospecific formation of tetrahedral intermediates. The results show that a large increase in the pKa of the active site histidine is not needed for the active site serine hydroxyl group to have an effective molarity of 6000.
2. Proteinase-catalyzed peptide synthesis in concentrated solutions of urea and other denaturing agents
V V Anisimova, I Y Filippova, E N Lysogorskaya, E S Oksenoit, S V Kolobanova, V M Stepanov Int J Pept Protein Res. 1996 Jan-Feb;47(1-2):28-35. doi: 10.1111/j.1399-3011.1996.tb00806.x.
Pepsin successfully catalyzed the synthesis of several hydrophobic octa- and decapeptides in dimethylformamide-water solutions containing concentrated urea at pH 4.65. The factors that influence peptide synthesis in the presence of urea were studied using condensation of the tripeptides Z-Ala-Ala-Phe-OH and H-Leu-Ala-Ala-OCH3 as a model. The dependence of Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 yield on pepsin concentration and pH, as well as the behavior of pepsin during peptide synthesis were studied. It was shown that pepsin catalyzed the synthesis of Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 in guanidine hydrochloride and sodium dodecyl sulfate solutions. Other proteinases, subtilisin and thermolysin, were applied for the synthesis of p-nitroanilides of tri- and tetrapeptides in urea solutions. Proteinase-catalyzed peptide synthesis in the presence of denaturing agents might help to overcome the limitations caused by poor solubility of the starting peptide derivatives, although this effect is sometimes counterbalanced by the product solubility.
3. Mechanism of action of papain: aryldehydroalanines as spectroscopic probes of acyl enzyme formation
M Smolarsky Biochemistry. 1978 Oct 31;17(22):4606-15. doi: 10.1021/bi00615a005.
The alpha,beta-unsaturated aromatic amino acids phenyldehydroalanine (PDA) and styryldehydroalanine (SDA) were synthesized and used as sensitive spectroscopic probes to study the acylation of papain by specific substrates and inhibitors. The spectral changes observed upon acylation of the enzyme with peptides containing these amino acids are large red shifts of the absorption maxima (lambda max) of the chromophores. The magnitudes of the absorption shifts were 49 nm (from 277 to 326 nm) for PDA peptide and 59 nm (from 318 to 377 nm) for SDA peptides. The following specific substrates were synthesized: Ac-Phe-Phe-PDA-OEt, Ac-Phe-PDA-NH2, Ala-Ala-Phe-SDA-OME, Ala-Ala-Phe-SDA-NH2, Lys-Ala-(o-benzyl)tyrosyl-SDA-OMe, and Lys-Ala-(o-benzyl)-tyrosyl-SDA-NH2. Similarly, the specific competitive inhibitors Ac-Phe-PDA (Ki = 5.3 X 10(-6) M), Z-Phe-SDA (Ki = 5.6 X 10(-5) M), Ala-Ala-Phe-SDA (Ki = 2.9 X 10(-5) M), and Lys-Ala-(o-benzyl)tyrosyl-SDA (Ki = 1.1 X 10(-5) M) were prepared. An additional chromophore was used to follow the noncovalent association of an inhibitor or substrate with papain, independently from the acylation or deacylation steps. This chromophore, which was introduced into the peptides at position P2, IS p-(p"-dimethylaminophenylazo) phenylalanine (DAP). The light absorption spectrum of DAP is dependent on its environment. The substrates Ala-Ala-DAP-SDA-OMe and Ala-Ala-DAP-SDA-NH2 and the competitive inhibitor Ala-Ala-DAP-SDA (Ki = 2.5 X 10(-6) M) were prepared. The noncovalent binding of these peptides to the active site of papain was followed either by the increase in the absorption at 480 nm or the decrease at 550 nm. With these petides the acylation and deacylation reactions could be followed simultaneously at 377 nm. The extent of acyl enzyme formation was found to decrease in a sigmoidal way with increasing pH, with a transition point around pH 5.5.
