Diazoacetyl norleucine methyl ester

Protease Classification. In order to rationally design an inhibitor for a protease it is first necessary to place it into one of four families of proteases (see Table V). For a new enzyme, a study of its inhibition profile with a series of general protease inhibitors is sufficient to classify it into one of the four families. The inhibitors usually used are diiso-propylphosphofluoridate (DFP) or phenylmethane sulfonyl fluoride (PMSF) for serine proteases, 1,10-phenanthroline for metalloproteases, thiol reagents such as iodoacetate or N-ethylmaleimide for thiol proteases, and pepstatin or diazo compounds such as diazoacetyl-norleucine methyl ester for carboxyl proteases. 

Table III. Acid Proteases Inhibited by Diazoacetyl Norleucine Methyl Ester (DAN)...

No selective irreversible inhibitors for carboxyl proteases have been developed yet. Reagents such as diazoacetyl-DL-norleucine methyl ester and l,2-epoxy-3-(-p-nitrophenoxyl) propane will inhibit many of the carboxyl proteases that have been examined, but little specificity is likely to be observed. 

IV. Aspartic proteases (acid proteases) are rare in Bacteria and contain one or more aspartic acid residues in their active center. Inactivation of the enzyme can be achieved by alkylation of the aspartic acid residues with DAN (diazoacetyl-DL-norleucine methyl ester) 91 . 

Renin can be inactivated more readily by aliphatic diazo compounds such as diazoacetyl-DL-norleucine methyl ester or diazoacelylglycine methyl ester in the presence of cupric ion. These reagents are soluble in water and hence do not require organic solvents. However, the reaction is less selective, since these latter reagents can react with other acid proteases. 

Chemical modifications of pepsin. Purified pepsin was subjected to treatment with the following previously investigated modifying agents (a) Diazoacetyl-D, L-norleucine methyl ester (24) (b) a-... 

Figure 1. The results are expressed as mean residue ellipti-cities in units of deg x cm x decimole . Protein samples were dissolved in 0.01 M sodium acetate, pH 4.0, at the following concentrations N-diazoacetyl-D, L-norleucine methyl ester-modified pepsin, 0.244 mg/ml l,2-epoxy-3-(p-nitrophenoxy)-propane-modified pepsin, 0.321 mg/ml hydroxamate-containing pepsin (4 mol/mole of pepsin), 0.227 mg/ml native pepsin, 0.364 mg/ml. The light pathlength was 10 mm for the near ultraviolet region (250-300 nm) and 1 mm for the far ultraviolet region (205-250 nm). Each curve represents an average of three measurements.

Figure 6. Inactivation of porcine renal renin by diazoacetyl-D,L-norleucine methyl ester (4 mM), in the presence ( ) and absence (0) of 2 mM cupric acetate at pH 6.2 and 14 . Ref (38)...

The submaxillary gland enzyme which had been completely inactivated by Cu -diazoacetyl-D,L-norleucine methyl ester was found to contain 1.2 moles of covalently bound norleucine per mole of enzyme upon amino acid analysis. This result suggested that an equimolar stoichiometric reaction between the catalytically essential group of the enzyme and the diazo inhibitor took place. 

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