title: Partial purification and characterization of two enzymes involved in isovaleric acid synthesis in Clostridium bifermentans.
abstract: Conversion of leucine to isovaleric acid by Clostridium bifermentans is achieved by the action of at least two enzymes. One is a transaminase producing alpha-ketoisocaproic acid, which was purified 30-fold from osmotic lysates of late-exponential phase cells by repeated chromatography on DEAE-Sepharose C16B and Sephacryl S300: this represented a 147-fold purification of activity found in sonically disrupted cells. This enzyme had an apparent molecular weight of approximately 190000 and was composed of six identically sized sub-units (molecular weight 31000 +/- 1000). Transamination required pyridoxal phosphate and pyruvate and was optimal at pH 8.6; the apparent Km for leucine was 7.0 mM. Activity was totally inhibited by 1 mM-p-chloromercuribenzoate and partially inhibited by other thiol reagents. The second enzyme decarboxylated alpha-ketoisocaproic acid to form isovaleric acid and was also partially purified by chromatography on DEAE-Sepharose C16B and Sephacryl S300. It has an apparent molecular weight of 240000 and required FAD and coenzyme A for activity; the Km for alpha-ketoisocaproic acid was 4.2 mM and activity was optimal around pH 8.0. This enzyme was a flavoprotein with absorption maxima at 280, 320 and 400 nm, and a fluorescent maximum at 500 nm. The prosthetic group, FAD, dissociated from the protein during purification resulting in an inactive apoenzyme which was only partially re-activated by FAD. Activity was completely inhibited by several thiol reagents tested at 1 mM.
mesh_terms: D002262:Carboxy-Lyases / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003013:Clostridium / Q000201:enzymology*; D003641:Deamination; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D007651:Keto Acids / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D008970:Molecular Weight; D010421:Pentanoic Acids / Q000096:biosynthesis*; D000637:Transaminases / Q000302:isolation & purification*; D014631:Valerates / Q000096:biosynthesis*
chemical_list: D045782:Hemiterpenes; D007651:Keto Acids; D010421:Pentanoic Acids; D014631:Valerates; D005182:Flavin-Adenine Dinucleotide; C008216:isovaleric acid; C013082:alpha-ketoisocaproic acid; D000637:Transaminases; D002262:Carboxy-Lyases; D007930:Leucine