Purification and properties of an acetoacetyl coenzyme A-reacting phosphotransbutyrylase from Clostridium beijerinckii (Clostridium butylicum) NRRL B593
- Virginia Polytechnic Institute and State Univ., Blacksburg (USA)
During the study of acetoacetyl coenzyme A (CoA)-reacting enzymes of Clostridium beijerinckii NRRL B593, a phosphate-dependent acetoacetyl-CoA-utilizing activity was detected in protein fractions devoid of thiolase and phosphotransacetylase. Further purification of this acetoacetyl-CoA-utilizing activity yielded an enzyme which may be designated as phosphotransbutyrylase (PTB; phosphate butyryltransferase (EC 2.3.1.19)). PTB from C. beijerinckii NRRL B593 was purified 160-fold with a yield of 14% and, with the best fractions, purified 190-fold to near homogeneity. It showed a native M{sub r} of 205,000 and a subunit M{sub r} of 33,000. PTB activity was sensitive to pH changes within the physiological range of 6 to 8. PTB exhibited a broad substrate specificity. The K{sub m} values at pH 7.5 for butyryl-CoA, acetoacetyl-CoA, and acetyl-CoA were 0.04, 1.10, and 3.33 mM, respectively. The V{sub max} values with butyryl-CoA and acetoacetyl-CoA were comparable, but the V{sub max}/K{sub m} was higher for butyryl-CoA than for acetoacetyl-CoA. An apparent K{sub m} of 6.5 mM for phosphate was obtained with butyryl-CoA as the cosubstrate, whereas it was 12.9 mM with acetoacetyl-CoA as the cosubstrate. It remains to be established whether the putative compound acetoacetyl phosphate is produced in the PTB-catalyzed reaction with acetoacetyl-CoA.
- DOE Contract Number:
- FG05-85ER13368
- OSTI ID:
- 6728144
- Journal Information:
- Applied and Environmental Microbiology; (USA), Vol. 56:3; ISSN 0099-2240
- Country of Publication:
- United States
- Language:
- English
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