a - D I A L K Y L A M I N O
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a-Dialkylamino Acid Transaminase from Pseudomonas cepacia. Purification, Crystallization, Physical, and Kinetic Properties* C. A. Lamartiniere,t Hajime Itoh,$ and Walter B. Dempseyi
a-Dialkylamino acid transaminase was purified from a strain of Pseudo/nonas cepuciu. The enzyme has been crystallized and subsequently characterized. By sedimentation equilibrium a molecular weight of 188,000 was determined for the native enzyme. A molecular weight of 47,000 (four subunits) was found when the enzyme was subjected t o strong dissociating agents (7 M guanidine hydrochloride-8 M urea), whereas amino acid analysis combined with tryptic peptide maps showed eight subunits. Electrophoresis of the crystalline ABsrRAc-T:
D
ecarboxylation-dependent transamination of a-dialkylamino acids was first demonstrated as a pyridoxal-catalyzed nonenzymatic reaction by Kalyankar and Snell (1962) in an extension of the pioneering studies of Herbst and Engel (1934). Later, an activity capable of catalyzing such a reaction was found in extracts of bacteria that had been isolated from enrichment cultures in which a-dialkylamino acids had served as the sole fixed nitrogen source (Aaslestad and Larson, 1964; Bailey and Dempsey, 1967; Dempsey, 1969). The activity has been purified t o apparent homogeneity from one such organism, and several of the enzymatic properties of the enzyme have been studied (Bailey and Dempsey, 1967; Bailey et a/., 1970). The physical properties of such a n enzyme have not been reported until now. The enzyme catalyzes the overall conversion of AIB’ and pyruvate to L-alanine, acetone, and carbon dioxide in two separate steps. In the first, AIB reacts with pyridoxal phosphate-enzyme t o form pyridoxamine phosphate-enzyme, COz, and acetone. In the second, a n exchange transamination occurs by which pyruvate is converted to L-alanine and pyridoxal phosphate-enzyme is regenerated. The enzyme is of interest, as has been pointed out earlier in greater detail (Bailey and Dempsey, 1967; Bailey e f al., 1970) because one active site cleaves two different types of groups attached t o a n cy-carbon atom of an amino acid. The original enzyme studied was isolated from a Pseudomonasfluorescens strain (Dempsey, 1969). In undertaking the study of the physical properties of this enzyme we found that the enzyme rapidly and irreversibly lost a large proportion of its activity during purification. After empirically determining * F r o m the Medical and Microbial Genetics Unit, V. A . Hospital and Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas 7 5 2 3 5 . Rereiced Airgrisf 2, 1971. This w o r k was supported in part by a training grant f r o m the U.S . Public Health Service 5T01 AM05567-03. t Present address: Physiologisch-Chemisches Institiit Der GeorgAugust Universitiit, G6ttingei1, Germany. $ Present address: Department of Biochemistry, Wakayania Medical University, 9, I
