A spectrophotometric rapid kinetic study of reactions catalysed by coenzyme-B12-dependent ethanolamine ammonia-lyase.

Abstract

Spectral changes during the transient states and the steady states of the reactions catalysed by the adenosylcobalamin‐dependent ethanolamine ammonia‐lyase with l‐2‐aminopropanol and 2‐aminoethanol as substrates were followed by using a rapid‐wavelength‐scanning, stopped‐flow spectrophotometer (800 spectra per second and each spectrum 340–560 nm). During the steady state of the reaction with the ‘poor’ substrate, l‐2‐aminopropanol (kcat∼ 1 s−1), under conditions of V, the spectrum of the coenzyme closely resembled that of cob(II)alamin. With the ‘good’ substrate, 2‐aminoethanol (kcat∼ 140 s−1), under the same conditions, the spectrum corresponded to a mixture of 58% cob(II)alamin and 42% cob(III)alamin. The latter may be either unchanged adenosylcobalamin or a coenzyme derivative with a covalent bond between a substrate carbon and the cobalt atom. The magnitude of a reported kinetic deuterium isotope effect favours the second possibility. There was no evidence for the presence of a cob(I)alamin intermediate. The transient phases of the reactions were dependent on the order of mixing of reactants. When enzyme was placed in one syringe of the stopped‐flow apparatus and coenzyme and substrate in the other, the cob(II)alamin intermediate formed slowly (first‐order rate constants of about 5 s−1 with l‐2‐aminopropanol as substrate and about 2 s−1 with 2‐aminoethanol). When the enzyme and coenzyme were placed in one syringe and the substrate in the other, the formation of the cob(II)alamin occurred much more rapidly (90 s−1 with l‐2‐aminopropanol and greater than 300 s−1 with 2‐aminoethanol). It is concluded that binding of adenosylcobalamin to the enzyme is followed by a slow change in the conformation of the enzyme molecule to give a catalytically active species (there is no observable change in the spectrum of the coenzyme concommitant with this process). The formation of active holoenzyme is slow on the time scale of subsequent catalytic steps when 2‐aminoethanol is the substrate. The spectra of the intermediates formed in the reactions with both substrates, the rates of their formation and their lifetimes allowed a detailed kinetic analysis of the reactions in terms of a three‐step mechanism involving binding of substrate, cob(II)alamin formation (k+2 step) and cob(II)alamin breakdown (k+3 step). In catalysis the difference between the substrates is expressed mainly in the k+3 step (240 s−1 and about 1 s−1 respectively for 2‐aminoethanol and l‐2‐aminopropanol, the respective k+2 values being 336 s−1 and 90 s−1).