Phe5(4-nitro)-bradykinin: a chromogenic substrate for assay and kinetics of the metalloendopeptidase meprin.
Phe5(4-nitro)-bradykinin has been identified as a good synthetic substrate to study the kinetics and mechanism of action of the metalloendopeptidase meprin. No convenient substrate for kinetic analysis of the enzyme had been previously described. HPLC analyses indicated that meprin cleaved bradykinin and nitrobradykinin between Phe5 (or Phe5(NO2)) and Ser6. Reaction rates for bradykinin were determined by quantitative HPLC analyses, whereas rates for nitrobradykinin were measured by continuous monitoring of the spectral change that occurs at 310 nm when the Phe(NO2)-Ser bond is hydrolyzed. For nitrobradykinin and unmodified bradykinin, respectively, Km values were 281 and 425 microM, kcat values were 28 and 22 s-1, and kcat/Km values were 9.7 x 10(4) and 5.1 x 10(4)M-1. The two products of bradykinin hydrolysis were not substrates for the enzyme, but they were inhibitors. The initial rates of hydrolysis of nitrobradykinin increased linearly with enzyme concentration (0.09-2.2 micrograms/ml), and increased linearly with temperature in the range from 15 to 55 degrees C. Hydrolysis of the substrate was optimal at alkaline pH values. The cysteine endopeptidases papain and cathepsin L and the metalloproteases thermolysin, angiotensin-converting enzyme, and neutral endopeptidase (EC 3.4.24.11) also cleaved nitrobradykinin, but at different peptide bonds than meprin. The single cleavage of nitrobradykinin at the Phe(NO2)-Ser bond and the concomitant spectral shift that occurs at alkaline pH makes this a particularly suitable substrate for meprin.
Wolz
R L
RL
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute & State University, Blacksburg 24061-0308.
Bond
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001920:Bradykinin / Q000031:analogs & derivatives* / Q000378:metabolism*; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000378:metabolism; D010206:Papain / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D013696:Temperature; D013820:Thermolysin / Q000378:metabolism; D008625:Tiopronin / Q000378:metabolism*
C068260:bradykinin, 4-nitro-Phe(5)-; D008625:Tiopronin; D002403:Cathepsins; D010450:Endopeptidases; D007703:Peptidyl-Dipeptidase A; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D010206:Papain; D015260:Neprilysin; D013820:Thermolysin; D001920:Bradykinin