Summary: Conversion of leucine to isovaleric acid by is achieved by the action of at least two enzymes. One is a transaminase producing α-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 for leucine was 7.0 mM. Activity was totally inhibited by 1 mM--chloromercuribenzoate and partially inhibited by other thiol reagents. The second enzyme decarboxylated α-ketoisocaproic acid to form isovaleric acid and was also partially purified by chromatography en DEAE-Sepharose C16B and Sephacryl S300. It has an apparent molecular weight of 240000 and required FAD and coenzyme A for activity; the for α-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.


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