Active site titration of bovine beta-trypsin by N alpha-(N,N-dimethylcarbamoyl)-alpha-aza-lysine p-nitrophenyl ester: kinetic and crystallographic analysis.
Kinetics of bovine beta-trypsin (trypsin) with the N alpha-(N,N-dimethylcarbamoyl)-alpha-aza-lysine p-nitrophenyl ester (Dmc-azaLys-ONp) was obtained at pH 6.2 and 21.0 degrees C. Dmc-azaLys-ONp shows the characteristics of an optimal active site titrant in that it (i) gives titrations in a short time, (ii) is a stable and soluble compound with a stoichiometric reaction that is easily and directly detectable, and (iii) allows titrations over a wide range of enzyme concentration. Moreover, the three-dimensional structure of the trypsin.N alpha-(N,N-dimet hylcarbamoyl)-alpha-aza-lysine acyl.enzyme adduct has been solved by X-ray crystallography at 2.0 A resolution (R = 0.145). The Dmc-azaLys moiety of the active site titrant is sited in the serine proteinase reaction center, and is covalently linked to the OG atom of the Ser195 catalytic residue.
Sartori
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Dipartimento di Genetica e Microbiologia, Universita di Pavia, Italy.
Djinovic Carugo
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Ferraccioli
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Balliano
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Milla
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Ascenzi
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Bolognesi
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M
D000818:Animals; D001372:Aza Compounds / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry; D017186:Titrimetry; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000378:metabolism*
D001372:Aza Compounds; D014361:Trypsin Inhibitors; C075756:N(alpha)(N,N-dimethylcarbamoyl)-alpha-azalysine 4-nitrophenyl ester; D012694:Serine; D014357:Trypsin; D008239:Lysine