N-acetylmuramoyl-L-alanine amidase assay based on specific radioactive labeling of muropeptide L-alanine: quantitation of the enzyme activity in the autolysin deficient Bacillus subtilis 168, flaD strain.
A sensitive and highly reproducible assay for N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) was devised, based on specific and homogeneous L-[14C]alanine labeling of the substrate, the peptidoglycan. The method involves partial purification of both the enzyme and the substrate and monitoring the muropeptide cleavage by coupling fluorodinitrobenzene to freed L-alanine NH2 groups. After acid hydrolysis of the substrate, the resulting DNP-L-alanine and L-alanine are separated by TLC, and radioactive counts in relevant spots are determined. Application of the method to the autolysin-endowed strain and an autolysis-deficient flaD-bearing mutant has revealed (i) that the N-acetylmuramoyl-L-alanine amidase behaves like an endoenzyme with an apparent Kcat(s-1) of 40, and (ii) that the residual enzyme activity in the flaD bearing strain amounts to 2.5 (+/- 0.1)% of that of the parent strain.
Margot
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P
Institut de Génétique et de Biologie Microbiennes, Lausanne, Switzerland.
Roten
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CA
Karamata
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D
D000409:Alanine / Q000378:metabolism; D000588:Amines / Q000032:analysis; D001412:Bacillus subtilis / Q000201:enzymology*; D002473:Cell Wall / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D004139:Dinitrofluorobenzene / Q000737:chemistry; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000032:analysis* / Q000378:metabolism; D010457:Peptidoglycan / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000032:analysis
D000588:Amines; D010457:Peptidoglycan; C028545:N-(2,4-dinitrophenyl)alanine; D010649:Phenylalanine; D004139:Dinitrofluorobenzene; D009238:N-Acetylmuramoyl-L-alanine Amidase; D000409:Alanine