Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans.
Cobyrinic acid a,c-diamide synthase, which catalyzes the conversion of cobyrinic acid to cobyrinic acid a,c-diamide via the intermediate formation of cobyrinic acid c-monoamide, was purified 155-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by high-performance liquid chromatography. The enzyme has an apparent molecular weight of 86,000 and consists of two identical subunits of Mr 45,000, as estimated by gel electrophoresis under denaturing conditions. Stepwise Edman degradation provided the N-terminal sequence of the first 15 amino acids. Glutamine was shown to be the preferred amino group donor (Km = 20.3 microM), but it could be replaced by ammonia (Km = 12 mM). The reaction was ATP dependent and exhibited a broad optimum pH around 7.3. Km values for (CN,aq)cobyrinic acid, (aq)2cobyrinic acid, and (CN,aq)cobyrinic acid c-monoamide were 160, greater than or equal to 250, and 71 microM, respectively. Hydrogenobyrinic acid and hydrogenobyrinic acid c-monoamide were shown to be much better substrates, with Km values of 0.41 and 0.21 microM, respectively.
Debussche
L
L
Département de Chimie Analytique, Centre de Recherche de Vitry, Rhône-Poulenc Santé, Vitry-sur-Seine, France.
Thibaut
D
D
Cameron
B
B
Crouzet
J
J
Blanche
F
F
D002845:Chromatography; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D000637:Transaminases / Q000302:isolation & purification* / Q000378:metabolism
D006882:Hydroxyapatites; D017886:Durapatite; D000637:Transaminases; C065829:cobyrinic acid a,c-diamide synthase