Steady-state and pre-steady state kinetic analysis of ornithine 4,5-aminomutase.

Abstract

Ornithine 4,5-aminomutase (4,5-OAM) is a pyridoxal 5'-phosphate and adenosylcobalamin-dependent enzyme that catalyzes a 1,2-rearrangement of the terminal amine of d-ornithine to form (2R, 4S)-diaminopentanoate. The gene encoding ornithine 4,5-aminomutase is clustered with other genes that function in the oxidative l-ornithine metabolic pathway present in a number of anaerobic bacteria. This chapter discusses the methodology for measuring 4,5-OAM activity using NAD+-dependent diaminopentanoate dehydrogenase, which functions downstream of 4,5-OAM in the l-ornithine metabolic pathway. The use of ornithine racemace, which functions upstream of 4,5-OAM, for the synthesis of d,l-ornithine-3,3,4,4,5,5-d6 is also presented. Finally, this chapter describes the anaerobic stopped-flow spectrophotometric analysis of 4,5-OAM. Information is provided on the integration of a stopped-flow system in the anaerobically-maintained glove, the preparation of anaerobic solutions, and the experimental approach.